Methods and devices for nucleic acid synthesis

ABSTRACT

Disclosed are devices and methods to synthesize polynucleotides and libraries of polynucleotides such as libraries of oligonucleotides. In exemplary embodiments, the device includes a support having a plurality of features. Each feature contains a plurality of oligonucleotides. Within each feature, each of the plurality of oligonucleotides includes an identical predetermined subunit sequence of X nucleosides and a degenerate sequence of Y nucleosides. A predetermined combination of a subset of the features can be used to produce a polynucleotide having a predetermined sequence of Z nucleosides.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 61/310,100, filed Mar. 3, 2010, theentire content of which is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

Devices and methods provided herein relate to the automated synthesis ofpolynucleotides and libraries of polynucleotides. More particularly, thedevices and methods are useful for performing synthesis ofoligonucleotides. In some embodiments, the devices provide a universal,combinatorial array for synthesizing oligonucleotides having any desiredsequence.

BACKGROUND

Synthetic biopolymers such as oligonucleotides play a pivotal role inmany fields such as molecular biology, forensic science, and medicaldiagnostics. Oligonucleotides, in particular, have become indispensabletools in modern biotechnology. Oligonucleotides are being used in a widevariety of techniques, ranging from diagnostic probing methods, PCR,antisense inhibition of gene expression to nucleic acid assembly. Thiswidespread use of oligonucleotides has led to an increasing demand forrapid, inexpensive and efficient methods for synthesizingoligonucleotides. Various types of microarray manufacturing devices andtechnologies have been described e.g. combinatorial array, ink-jetting,direct surface printing approaches have been extensively described. Mostof theses technologies use numerous valves and tubes and other fluidhandling components. As such a need remains for a quick, cost effective,scalable oligonucleotide synthesis device able to generate high qualityoligonucleotides suitable for an array of applications.

SUMMARY OF THE INVENTION

Aspects of the technology provided herein relate to devices and methodsfor synthesizing polynucleotides. Aspects of the invention relate todevices and methods for the synthesis of a plurality of polynucleotidesand/or libraries of polynucleotides on a solid support. In one aspect ofthe invention, a device for synthesizing at least one polynucleotidehaving a predetermined sequence is provided. The device can include asupport having a plurality of features, each feature having a pluralityof oligonucleotides, and within each feature each of the plurality ofoligonucleotides including an identical predetermined subunit sequenceof X nucleosides and a degenerate sequence of Y nucleosides. In someembodiments, X is between 2 and 50 nucleosides. More particularly, X isbetween 3 and 20 nucleosides. In some examples, X is 3, 4, 5, 6, 7, 8,9, or 10 nucleosides. In certain embodiments, Y is between 5 and 100nucleosides. More particularly, Y is between 5 and 20, or Y is 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In someembodiments, the device can have at least 4^(X) different features. Insome examples, the device has at least 100, 1,000, 4,000, 10,000 or moredifferent features. The predetermined subunit sequences can be differentbetween the features. A subset of the plurality of the features togethercan represent the polynucleotide having the predetermined sequence of Znucleosides. In certain embodiments, at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, 99% or more of all possible combinations(4^(Z)) of the polynucleotide having the predetermined sequence of Znucleosides are represented on a single device.

Another aspect of the invention relates to a device for synthesizing atleast one polynucleotide having a predetermined sequence on a solidsupport. In some embodiments, the predetermined sequence includes asubunit sequence. The solid support can have a plurality of spots, andeach of the plurality of spots includes a plurality of oligonucleotideshaving a predetermined subunit sequence. In some embodiments, theplurality of oligonucleotides are covalently linked at their 3′ end viaa plurality of binding sequences on the solid support. The devicefurther can include a microfluidic member for providing a droplet to afirst spot having a first oligonucleotide having a first predeterminedsubunit sequence to substantially cover the first spot, and the dropletcan include one or more reagents that allow one or more of annealing,denaturing, chain extension reaction, ligation, and digestion reactionto produce a first product which includes the first predeterminedsubunit sequence. The device can also include a member for advancingmicrofluidic communication between the first spot and a second spot. Thesecond spot has a second predetermined subunit sequence, and at thesecond spot one or more of annealing, denaturing, chain extensionreaction, ligation, and digestion is allowed to produce a second productthat includes the first and the second predetermined subunit sequences.

In certain embodiments, the plurality of binding sequences aredegenerate sequences having a length of N1 nucleosides, and include upto 4^(N1) different sequences within a single spot or feature. In someembodiments, N1 is between 5 and 100 nucleosides. In some embodiments,N1 is between 10 and 50 nucleosides. In some examples, N1 is 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In oneexample, N1 is 10 nucleosides.

In some embodiments, the subunit sequence has a length of N2 and is oneof 4^(N2) possible sequences. The solid support includes 4^(N2) spots ora subset or superset thereof. In some embodiments, N2 is between 2 and50. In some embodiments, N2 is between 4 and 20. In some examples, N2 is3, 4, 5, 6, 7, 8, 9, or 10. In one example, N2 is 5.

In some embodiments, the device further includes means for controlling atemperature of the droplet at each of the plurality of spots on thesolid support. In certain embodiments, the temperature is controlled atabove a predetermined temperature which corresponds to an averageannealing temperature of the plurality of binding sequences. In someexamples, the temperature is controlled at up to 20° C. above thepredetermined temperature. In certain examples, the temperature iscontrolled at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10° C. above thepredetermined temperature. In some embodiments, the temperature iscontrolled at above the predetermined temperature such that duplexes,stabilized by a binding agent, remain substantially annealed at saidtemperature. The binding agent can be a polymerase or a subunit thereof.

In certain embodiments, the droplet contains a primer that at leastpartially binds to at least one of the plurality of the bindingsequences on the first spot, a polymerase or a subunit thereof, anddNTPs or an analog thereof, thereby allowing chain extension off of theprimer using the first predetermined subunit sequence as template toproduce the first product comprising the first predetermined subunitsequence. The primer can be a plurality of degenerate primers. The firstproduct can serve as a primer for chain extension at the second spotwhich uses the second predetermined subunit sequence as template toproduce the second product comprising the first and the secondpredetermined subunit sequences.

In some embodiments, the first and the second predetermined subunitsequences are the same or complementary.

In some embodiments, the plurality of oligonucleotides at the 3′ end arecovalently linked to the plurality of binding sequences via a linkersequence.

A further aspect of the invention includes a device for synthesizing atleast one polynucleotide having a predetermined sequence. In someembodiments, the predetermined sequence includes a subunit sequence. Thesolid support can have a plurality of spots, and each of the pluralityof spots can include a plurality of oligonucleotides having apredetermined subunit sequence. The plurality of oligonucleotides arecovalently linked at their 3′ end via a plurality of binding sequenceson the solid support. The plurality of binding sequences can bedegenerate sequences having a length of N1 nucleosides and can compriseup to 4^(N1) different sequences on the entire device. The devicefurther includes a member for providing a solution to a spot having afirst oligonucleotide having a first predetermined subunit sequence. Insome embodiment, the spot is substantially covered by the solution. Thesolution can include one or more reagents that allow one or more ofannealing, denaturing, chain extension reaction, ligation, and digestionreaction to produce a product comprising the predetermined subunitsequence.

Other aspects of the invention relate to methods for synthesizing atleast one polynucleotide having a predetermined sequence. Thepolynucleotide can comprise a subunit sequence. In some embodiments, themethod includes (a) providing a solid support having a plurality ofspots thereon, wherein each of the plurality of spots comprises aplurality of oligonucleotides having a predetermined subunit sequence,wherein the plurality of oligonucleotides are covalently linked at the3′ end via a plurality of binding sequences on the solid support; (b)providing a droplet to a first spot having a first oligonucleotidehaving a first predetermined subunit sequence, wherein the dropletcomprises one or more reagents that allow one or more of annealing,denaturing, chain extension reaction, ligation, and digestion reactionto produce a first product comprising the first predetermined subunitsequence, and (c) advancing microfluidic communication between the firstspot and a second spot having a second oligonucleotide having a secondpredetermined subunit sequence, whereby allowing one or more ofannealing, denaturing, chain extension reaction, ligation, and digestionreaction at the second spot to produce a second product comprising thefirst and the second predetermined subunit sequences.

The method can further include repeating step (c) until thepredetermined sequence having the desired subunit sequences is produced.In certain embodiments, the plurality of binding sequences comprisesdegenerate sequences having a length of N1 nucleosides, and theplurality of binding sequences comprise up to 4^(N1) differentsequences. In some embodiments, N1 is between 5 and 100. In someembodiments, N1 is between 10 and 50 nucleosides. In some examples, N1is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nucleosides. In one example, N1 is 10 nucleosides. In some embodiments,the subunit sequence has a length of N2 and is one of 4^(N2) possiblesequences. The solid support can include 4^(N2) spots or a subset orsuperset thereof. In some embodiments, N2 is between 2 and 50nucleosides. In some embodiments, N2 is between 4 and 20 nucleosides. Insome examples, N2 is 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides. In oneexample, N2 is 5 nucleosides.

In some embodiments, the method further includes controlling atemperature of the droplet at each of the plurality of spots on thesolid support. The temperature can be controlled at above apredetermined temperature which corresponds to an average annealingtemperature of the plurality of binding sequences. In some embodiments,the temperature is controlled at up to 20° C. above the predeterminedtemperature. In some examples, the temperature is controlled at 1, 2, 3,4, 5, 6, 7, 8, 9, or 10° C. above the predetermined temperature. Incertain examples, the temperature is controlled at above thepredetermined temperature such that duplexes stabilized by a bindingagent remain substantially annealed at said temperature. In one example,the binding agent is a polymerase or a subunit thereof.

In some embodiments, the method further includes providing in thedroplet a primer that at least partially binds to at least one of theplurality of the binding sequences on the first spot, a polymerase or asubunit thereof, and dNTPs or analogs thereof, thereby allowing chainextension of the primer using the first predetermined subunit sequenceas a template to produce the first product comprising the firstpredetermined subunit sequence. In some examples, the primer comprises aplurality of degenerate primers.

In various embodiments, the method further includes cleaving the primerto remove unwanted primer sequences from the predetermined sequence. Incertain embodiments, the predetermined sequence is amplified via chainextension reactions.

In some embodiments, the method further includes using the first productas a primer for chain extension at the second spot and using the secondpredetermined subunit sequence as a template to produce the secondproduct comprising the first and the second predetermined subunitsequences.

In various embodiments, one or more of the steps of the method arerepeated until an desired amount of the predetermined sequence havingthe desired subunit sequences is produced.

In another aspect, a method is provided for synthesizing apolynucleotide having a predetermined sequence. The polynucleotide cancomprise a subunit sequence. The method includes (a) providing a solidsupport having a plurality of spots thereon, wherein each of theplurality of spots comprises a plurality of oligonucleotides having apredetermined subunit sequence, wherein the plurality ofoligonucleotides at the 3′ end are covalently linked via a plurality ofbinding sequences on the solid support, wherein the plurality of bindingsequences are degenerate sequences having a length of N1 nucleosides andcomprise up to 4^(N1) different sequences; (b) providing a firstsolution to a first spot having a first plurality of oligonucleotideshaving a first predetermined subunit sequence, wherein the firstsolution comprises one or more reagents that allow one or more ofannealing, denaturing, chain extension, ligation, and digestion reactionto produce a first product comprising the first predetermined subunitsequence; and (c) providing a second solution to a second spot having asecond plurality of oligonucleotides having a second predeterminedsubunit sequence, wherein the second solution comprises one or morereagents that allow one or more of annealing, denaturing, chainextension reaction, ligation, and digestion reaction to produce a secondproduct comprising the second predetermined subunit sequence. In someembodiment, the first solution and the second solution coversubstantially the first and second spot, respectively.

In various embodiments, the first and the second predetermined subunitsequences are different. In some embodiments, the first product servesas a primer for chain extension at the second spot which uses the secondpredetermined subunit sequence as a template to produce the secondproduct comprising the first and the second predetermined subunitsequences. In certain embodiments, the first and the secondpredetermined subunit sequences are the same or complementary, and themethod further comprises combining the first product and the secondproduct and amplifying therefrom.

Other features and advantages of the devices and methods provided hereinwill be apparent from the following detailed description, and from theclaims. The claims provided below are hereby incorporated into thissection by reference. The various embodiments described herein can becomplimentary and can be combined or used together in a mannerunderstood by the skilled person in view of the teachings containedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary method for the elongation ofpolynucleotides on a solid support using repeated polymerase extensionreactions.

FIG. 2A illustrates an exemplary device having a feature (210) whichcomprises a plurality of oligonucleotides (201, SEQ ID NO: 1) having adegenerate binding sequence (203) and a payload sequence (202). FIG. 2Billustrates under an exemplary stringent melt condition, stabilizedduplex (220) proceeding to chain extension while non-stabilized duplex(240) does not.

FIG. 3 illustrates an exemplary universal DNA oligomer array.

FIG. 4A illustrates an exemplary binding of a constructionsingle-stranded DNA to an oligonucleotide immobilized (SEQ ID NO: 2) onan array. FIG. 4B illustrates an exemplary binding of a constructionsingle-stranded DNA to an oligonucleotide immobilized (SEQ ID NO. 2) onan array in a region other than the 3′ end (n1-n6).

FIG. 5 illustrates the base addition to the construction single-strandedDNA (SEQ ID NO: 3).

FIGS. 6 and 7 illustrate one embodiment for synthesizing a DNA constructfrom a universal oligomer array. FIG. 6A illustrates a DNA arraycontaining all possible M-mers in N spots according to a non-limitingembodiment. FIG. 6B illustrates the hybridization of a universaldigestable primer P_(d) to the universal priming sites P_(d) on thearray according to a non-limiting embodiment. FIG. 7A illustratesextension of the primers P_(d) extended using a polymerase and dNTPs.FIG. 7B illustrate digestion of the digestable primers P_(d) accordingto a non-limiting embodiment.

FIG. 8 illustrates an universal DNA array being processed usingmicrofluidic and laser devices.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the technology provided herein are useful for increasing theaccuracy, yield, throughput, and/or cost efficiency of nucleic acidsynthesis and assembly reactions. As used herein the terms “nucleicacid”, “polynucleotide”, “oligonucleotide” are used interchangeably andrefer to naturally-occurring or synthetic polynucleotide forms ofnucleotides. The oligonucleotides and nucleic acid molecules of thepresent invention can be formed from naturally occurring nucleotides,for example forming deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) molecules. Alternatively, the naturally occurring oligonucleotidescan include structural modifications to alter their properties, such asin peptide nucleic acids (PNA) or in locked nucleic acids (LNA). Thesolid phase synthesis of oligonucleotides and nucleic acid moleculeswith naturally occurring or artificial bases is well known in the art.The terms should be understood to include equivalents, analogs of eitherRNA or DNA made from nucleotide analogs and as applicable to theembodiment being described, single-stranded or double-strandedpolynucleotides. Nucleotides useful in the invention include, forexample, naturally-occurring nucleotides (for example, ribonucleotidesor deoxyribonucleotides), or natural or synthetic modifications ofnucleotides, or artificial bases.

A polymer refers to at least two monomers that are linked together.Biopolymer refers to a polymer found in biological systems comprising aplurality of biological monomeric units or biomonomers linked together,such as nucleic acids (including DNA, RNA, polynucleotides,oligonucleotides, oligonucleotide probes) sugars, proteins, antibodies,antigens, enzymes, coenzymes, ligands, receptors, hormones and labels,and genes that specify any of the above. Biopolymers include compoundscomposed of or containing amino acid or nucleotide analogs ornon-nucleotide groups. This includes polynucleotides in which theconventional backbone has been replaced with a non-naturally occurringor synthetic backbone, and nucleic acids in which one or more of theconventional bases has been replaced with a synthetic base capable ofparticipating in Watson-Crick type hydrogen bonding interactions. Asused herein, the term monomer refers to a member of a set of smallmolecules which are and can be joined together to from an oligomer, apolynucleotide or a compound composed of two or more members. Theparticular ordering of monomers within a polynucleotide is referred toherein as the “sequence” of the polynucleotide. The set of monomersincludes but is not limited to example, the set of common L-amino acids,the set of D-amino acids, the set of synthetic and/or natural aminoacids, the set of nucleotides and the set of pentoses and hexoses.Aspects of the invention described herein primarily with regard to thepreparation of oligonucleotides, but could readily be applied in thepreparation of other polynucleotides such as peptides or polypeptides,polysaccharides, phospholipids, heteropolynucleotides, polyesters,polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, or any otherpolynucleotides.

As used herein, the term “predetermined sequence” means that thesequence of the polynucleotide is known and chosen before synthesis orassembly of the polynucleotide. In particular, aspects of the inventionis described herein primarily with regard to the preparation of nucleicacids molecules, the sequence of the oligonucleotide or polynucleotidebeing known and chosen before the synthesis or assembly of the nucleicacid molecules. In some embodiments of the technology provided herein,immobilized oligonucleotides or polynucleotides are used as a source ofmaterial. In various embodiments, the methods described herein useoligonucleotides, their sequence being determined based on the sequenceof the final polynucleotides constructs to be synthesized. In oneembodiment, oligonucleotides are short nucleic acid molecules. Forexample, oligonucleotides can be from 2 to about 10 nucleotides, from 10to about 30 nucleotides, from 30 to about 50 nucleotides, from 50 toabout 100 nucleotides, or more than about 100 nucleotides long. However,shorter or longer oligonucleotides can be used. Oligonucleotides can bedesigned to have different length.

Oligonucleotides or polynucleotides of any length can be produced by thedevices and methods described herein. In some embodiments, the sequenceof the desired oligonucleotide or polynucleotide constructs can bedivided up into a plurality of shorter sequences that can be synthesizedin parallel and/or assembled into a single or a plurality of desiredoligonucleotide or polynucleotide constructs using the methods describedherein. In some embodiments, the synthesis and/or assembly procedure caninclude several parallel and/or sequential reaction steps in which aplurality of different nucleic acids or oligonucleotides are synthesizedor immobilized, primer-extended, and are combined in order to beassembled (e.g., by extension or ligation as described herein) togenerate a longer nucleic acid product to be used for further assembly,cloning, or other applications (see U.S. provisional Application No.61/235,677 and PCT Application No. PCT/US09/55267, both of which areincorporate herein by reference in their entirety).

Aspects of the invention relate to devices and/or methods forsynthesizing at least one polynucleotide having a desired orpredetermined sequence on a solid support. The device described hereinpermits relatively inexpensive, rapid, and high fidelity construction ofessentially any polynucleotide desired. Unless defined otherwise below,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. Still, certain elements are defined herein for thesake of clarity. It must be noted that, as used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the context clearly dictates otherwise. Thus,for example, reference to “a polynucleotide” can include more than onepolynucleotide.

In one aspect, a device for synthesizing a polynucleotide having apredetermined sequence is provided. The device can include a supporthaving a plurality of features, each feature having a plurality ofoligonucleotides, and within each feature each of the plurality ofoligonucleotides includes an identical predetermined subunit sequence ofX nucleosides and a degenerate sequence of Y nucleosides. In someembodiments, X is between 2 and 50. More particularly, X is between 3and 20. In some examples, X is 3, 4, 5, 6, 7, 8, 9, or 10. In certainembodiments, Y is between 5 and 100. More particularly, Y is between 5and 20, or Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20. In some embodiments, the device can have at least 4^(X) differentfeatures. In some examples, the device has at least 100, 1,000, 4,000,10,000 or more different features. In some embodiments, thepredetermined subunit sequences can be different between each of thefeatures. Yet in other embodiments, the, the predetermined subunitsequences can be the same. In some embodiments, a subset of theplurality of the features together can represent the polynucleotidehaving the predetermined sequence of Z nucleosides. In certainembodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,98%, 99% or more of all possible combinations (4^(Z)) of thepolynucleotide having the predetermined sequence of Z nucleosides arerepresented on a single device. In some embodiments, any predeterminedcombination of a subset of the features can be used to produce apolynucleotide of Z nucleosides, such that the device represents atleast 4^(Z) different polynucleotides.

Another aspect of the invention relates to a device for synthesizing atleast one polynucleotide having a predetermined sequence on a solidsupport. The predetermined sequence includes a subunit sequence. Thesolid support has a plurality of spots, and each of the plurality ofspots includes a plurality of oligonucleotides having a predeterminedsubunit sequence. The plurality of oligonucleotides are covalentlylinked at their 3′ end via a plurality of binding sequences on the solidsupport. The device further includes a member for providing a droplet toa first spot having a plurality of first oligonucleotides having a firstpredetermined subunit sequence. In some embodiments, the droplet cansubstantially cover the first spot. The droplet can include one or morereagents that allow one or more of annealing, denaturing, chainextension reaction, ligation, and digestion reaction to produce a firstproduct which includes the first predetermined subunit sequence. Thedevice also includes a member for advancing microfluidic communicationbetween the first spot and a second spot. In some embodiments, thesecond spot has a second plurality of oligonucleotides having a secondpredetermined subunit sequence, and at the second spot one or more ofannealing, denaturing, chain extension reaction, ligation, and digestionis allowed to produce a second product that includes the first and thesecond predetermined subunit sequences.

A further aspect of the invention includes a device for synthesizing atleast one polynucleotide having a predetermined sequence. Thepredetermined sequence can include a subunit sequence. In someembodiments, the solid support has a plurality of spots, and each of theplurality of spots includes a plurality of oligonucleotides having apredetermined subunit sequence. The plurality of oligonucleotides arecovalently linked at their 3′ end via a plurality of binding sequenceson the solid support. In some embodiments, the plurality of bindingsequences comprises degenerate sequences having a length of N1nucleosides and comprises up to 4^(N1) different sequences on the entiredevice. The device further includes a member for providing a solution toa spot having a predetermined subunit sequence to substantially coverthe spot. The solution includes one or more reagents that allow one ormore of annealing, denaturing, chain extension reaction, ligation, anddigestion reaction to produce a product comprising the predeterminedsubunit sequence.

Other aspects of the invention relate to methods for synthesizing atleast one polynucleotide having a predetermined sequence. In someembodiments, the at least one polynucleotide sequence comprises asubunit sequence. In some embodiments, the method includes:

(a) providing a solid support having a plurality of spots thereon,wherein each of the plurality of spots comprises a plurality ofoligonucleotides having a predetermined subunit sequence, wherein theplurality of oligonucleotides are covalently linked at the 3′ end via aplurality of binding sequences on the solid support;

(b) providing a droplet to a first spot having a first plurality ofoligonucleotides having a first predetermined subunit sequence, whereinthe droplet comprises one or more reagents that allow one or more ofannealing, denaturing, chain extension reaction, ligation, and digestionreaction to produce a first product comprising the first predeterminedsubunit sequence; and

(c) advancing microfluidic communication between the first spot and asecond spot having a second plurality of oligonucleotides having asecond predetermined subunit sequence, whereby allowing one or more ofannealing, denaturing, chain extension reaction, ligation, and digestionreaction at the second spot to produce a second product comprising thefirst and the second predetermined subunit sequences. In someembodiments, the method comprise providing a first droplet at the firstspot and second droplet at the second spot to cover substantially thefirst and the second spot respectively.

In another aspect, a method is provided for synthesizing at least onepolynucleotide having a predetermined sequence. The method includes:

(a) providing a solid support having a plurality of spots thereon,wherein each of the plurality of spots comprises a plurality ofoligonucleotides having a predetermined subunit sequence, wherein theplurality of oligonucleotides are covalently linked at the 3′ end via aplurality of binding sequences on the solid support, wherein theplurality of binding sequences are degenerate sequences having a lengthof N1 nucleosides and comprise up to 4^(N1) different sequences;

(b) providing a first solution to a first spot having a first pluralityof oligonucleotuides having a first predetermined subunit sequence tosubstantially cover the first spot, wherein the first solution comprisesone or more reagents that allow one or more of annealing, denaturing,chain extension reaction, ligation, and digestion reaction to produce afirst product comprising the first predetermined subunit sequence; and

(c) providing a second solution to a second spot having a secondplurality of oligonucleotides having a second predetermined subunitsequence to substantially cover the second spot, wherein the secondsolution comprises one or more reagents that allow one or more ofannealing, denaturing, chain extension reaction, ligation, and digestionreaction to produce a second product comprising the second predeterminedsubunit sequence.

In various embodiments, at least 1,000, at least 10,000, at least20,000, or at least 30,000 different polynucleotides are synthesized inparallel or sequentially using the device and/or method of theinvention. The solid support can be a pin, a rod, a cylinder, a tube, astrip, a slide or the like. Polynucleotides can be synthesized on theentire solid support surface. In some embodiments, polynucleotides canbe synthesized on part of the solid support surface in a synthesis area.

In various embodiments, the synthesis area is made of a porous materialor of particles of solid material such as beads. The solid support canbe composed of glass, silica, plastic, ceramic, beads, metal, organicmaterial, nylon, semiconductor material, oxides or combinations thereofor can be an optical fiber. In some embodiments, the solid supportcomprises a core member and a synthesis solid surface covering a sectionof the core member on which polynucleotides are synthesized. Thesynthesis solid surfaces are interlocked onto the core members viamagnetic, mechanical, electrical or chemical mechanisms. In someembodiments, the solid support is magnetized or axially magnetized. Thesolid supports can be moved independently by electrical, magnetic,electro-magnetic, pneumatic, mechanical means or any combinationthereof. In a preferred embodiment, the solid supports are movedvertically in relation to the holder and independently of each other.

The synthesis of polynucleotides involves the creation of chemicallinkages between monomers. Accordingly, the synthesis ofoligonucleotides involves the creation of phosphodiester bonds. Thecreation of phosphodiester bonds involves a number of sequentialchemical steps that can be divided into a number of physical steps. Insome embodiments, the different chemical steps are carried outsequentially at the different features or spots designated by thedevice. The features or spots can be arranged linearly or circularly.Each of these features or spots can have access to a reagent forperforming a step of said polynucleotides synthesis. For example, foroligonucleotide synthesis, the features or spots can be accessed by washreagents, annealing buffer, denaturing buffer, chain extension reactionreagents, ligation reagents, and digestion reagents.

Some aspects of the invention relate to a polynucleotidesynthesis/assembly process wherein synthetic oligonucleotides aredesigned and used as templates for primer extension reactions to producepolynucleotides of desired sequence. During enzymatic amplification orchain extension reactions, an initial step involves the non-covalentbinding between a primer sequence and a template sequence to for aduplex. The term “duplex” refers to a nucleic acid molecule that is atleast partially double-stranded. A “stable duplex” refers to a duplexthat is relatively more likely to remain hybridized to a complementarysequence under a given set of hybridization conditions. In an exemplaryembodiment, a stable duplex refers to a duplex that does not contain abase pair mismatch, insertion, or deletion. An “unstable duplex” refersto a duplex that is relatively less likely to remain hybridized to acomplementary sequence under a given set of hybridization conditionssuch as stringent melt. In an exemplary embodiment, an unstable duplexrefers to a duplex that is not 100% double-stranded within targetsequences, e.g., due to lack of stabilizing agent (e.g., a polymerase ora subunit thereof) under stringent conditions.

Solid Supports

In some embodiments, the methods and provided herein useoligonucleotides that are immobilized on a surface or substrate (e.g.,support-bound oligonucleotides). As used herein the term “support” and“substrate” are used interchangeably and refers to a porous ornon-porous solvent insoluble material on which polymers such as nucleicacids are synthesized or immobilized. As used herein “porous” means thatthe material contains pores having substantially uniform diameters (forexample in the nm range). Porous materials include paper, syntheticfilters etc. In such porous materials, the reaction may take placewithin the pores. The support can have any one of a number of shapes,such as pin, strip, plate, disk, rod, bends, cylindrical structure,particle, including bead, nanoparticles and the like. The support canhave variable widths. The support can be hydrophilic or capable of beingrendered hydrophilic and includes inorganic powders such as silica,magnesium sulfate, and alumina; natural polymeric materials,particularly cellulosic materials and materials derived from cellulose,such as fiber containing papers, e.g., filter paper, chromatographicpaper, etc.; synthetic or modified naturally occurring polymers, such asnitrocellulose, cellulose acetate, poly (vinyl chloride),polyacrylamide, cross linked dextran, agarose, polyacrylate,polyethylene, polypropylene, poly (4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF) membrane, glass, controlledpore glass, magnetic controlled pore glass, ceramics, metals, and thelike; either used by themselves or in conjunction with other materials.

In some embodiments, oligonucleotides are synthesized on an arrayformat. For example, single-stranded oligonucleotides are synthesized insitu on a common support wherein each oligonucleotide is synthesized ona separate or discrete feature (or spot) on the substrate. In preferredembodiments, single stranded oligonucleotides are bound to the surfaceof the support or feature. As used herein the term “array” refers to anarrangement of discrete features for storing, routing, amplifying andreleasing oligonucleotides or complementary oligonucleotides for furtherreactions. In a preferred embodiment, the support or array isaddressable: the support includes two or more discrete addressablefeatures at a particular predetermined location (i.e., an “address”) onthe support. Therefore, each oligonucleotide molecule of the array islocalized to a known and defined location on the support. The sequenceof each oligonucleotide can be determined from its position on thesupport. Moreover, addressable supports or arrays enable the directcontrol of individual isolated volumes such as droplets. In someembodiments, the size of the defined feature is chosen to allowformation of a microvolume droplet on the feature, each droplet beingkept separate from each other. As described herein, features aretypically, but need not be, separated by interfeature spaces to ensurethat droplets between two adjacent features do not merge. Interfeatureswill typically not carry any oligonucleotide on their surface and willcorrespond to inert space. In some embodiments, features andinterfeatures may differ in their hydrophilicity or hydrophobicityproperties. In some embodiments, features and interfeatures may comprisea modifier as described herein.

In some embodiments, oligonucleotides are attached, spotted,immobilized, surface-bound, supported or synthesized on the discretefeatures of the surface or array. Oligonucleotides may be covalentlyattached to the surface or deposited on the surface. Arrays may beconstructed, custom ordered or purchased from a commercial vendor (e.g.,Agilent, Affymetrix, Nimblegen). Various methods of construction arewell known in the art e.g., maskless array synthesizers, light directedmethods utilizing masks, flow channel methods, spotting methods etc. Insome embodiments, construction and/or selection oligonucleotides may besynthesized on a solid support using maskless array synthesizer (MAS).Maskless array synthesizers are described, for example, in PCTapplication No. WO 99/42813 and in corresponding U.S. Pat. No.6,375,903. Other examples are known of maskless instruments which canfabricate a custom DNA microarray in which each of the features in thearray has a single-stranded DNA molecule of desired sequence.

Other methods for synthesizing construction and/or selectionoligonucleotides include, for example, light-directed methods utilizingmasks, flow channel methods, spotting methods, pin-based methods, andmethods utilizing multiple supports. Light directed methods utilizingmasks (e.g., VLSIPS™ methods) for the synthesis of oligonucleotides isdescribed, for example, in U.S. Pat. Nos. 5,143,854, 5,510,270 and5,527,681. These methods involve activating predefined regions of asolid support and then contacting the support with a preselected monomersolution. Selected regions can be activated by irradiation with a lightsource through a mask much in the manner of photolithography techniquesused in integrated circuit fabrication. Other regions of the supportremain inactive because illumination is blocked by the mask and theyremain chemically protected. Thus, a light pattern defines which regionsof the support react with a given monomer. By repeatedly activatingdifferent sets of predefined regions and contacting different monomersolutions with the support, a diverse array of polymers is produced onthe support. Other steps, such as washing unreacted monomer solutionfrom the support, can be optionally used. Other applicable methodsinclude mechanical techniques such as those described in U.S. Pat. No.5,384,261.

Additional methods applicable to synthesis of construction and/orselection oligonucleotides on a single support are described, forexample, in U.S. Pat. No. 5,384,261. For example, reagents may bedelivered to the support by either (1) flowing within a channel definedon predefined regions or (2) “spotting” on predefined regions. Otherapproaches, as well as combinations of spotting and flowing, may beemployed as well. In each instance, certain activated regions of thesupport are mechanically separated from other regions when the monomersolutions are delivered to the various reaction sites. Flow channelmethods involve, for example, microfluidic systems to control synthesisof oligonucleotides on a solid support. For example, diverse polymersequences may be synthesized at selected regions of a solid support byforming flow channels on a surface of the support through whichappropriate reagents flow or in which appropriate reagents are placed.Spotting methods for preparation of oligonucleotides on a solid supportinvolve delivering reactants in relatively small quantities by directlydepositing them in selected regions. In some steps, the entire supportsurface can be sprayed or otherwise coated with a solution, if it ismore efficient to do so. Precisely measured aliquots of monomersolutions may be deposited dropwise by a dispenser that moves fromregion to region.

Pin-based methods for synthesis of oligonucleotides on a solid supportare described, for example, in U.S. Pat. No. 5,288,514. Pin-basedmethods utilize a support having a plurality of pins or otherextensions. The pins are each inserted simultaneously into individualreagent containers in a tray. An array of 96 pins is commonly utilizedwith a 96-container tray, such as a 96-well microtiter dish. Each trayis filled with a particular reagent for coupling in a particularchemical reaction on an individual pin. Accordingly, the trays willoften contain different reagents. Since the chemical reactions have beenoptimized such that each of the reactions can be performed under arelatively similar set of reaction conditions, it becomes possible toconduct multiple chemical coupling steps simultaneously.

In another embodiment, a plurality of oligonucleotides may besynthesized on multiple supports. One example is a bead based synthesismethod which is described, for example, in U.S. Pat. Nos. 5,770,358;5,639,603; and 5,541,061. For the synthesis of molecules such asoligonucleotides on beads, a large plurality of beads is suspended in asuitable carrier (such as water) in a container. The beads are providedwith optional spacer molecules having an active site to which iscomplexed, optionally, a protecting group. At each step of thesynthesis, the beads are divided for coupling into a plurality ofcontainers. After the nascent oligonucleotide chains are deprotected, adifferent monomer solution is added to each container, so that on allbeads in a given container, the same nucleotide addition reactionoccurs. The beads are then washed of excess reagents, pooled in a singlecontainer, mixed and re-distributed into another plurality of containersin preparation for the next round of synthesis. It should be noted thatby virtue of the large number of beads utilized at the outset, therewill similarly be a large number of beads randomly dispersed in thecontainer, each having a unique oligonucleotide sequence synthesized ona surface thereof after numerous rounds of randomized addition of bases.An individual bead may be tagged with a sequence which is unique to thedouble-stranded oligonucleotide thereon, to allow for identificationduring use.

Pre-synthesized oligonucleotide and/or polynucleotide sequences may beattached to a support or synthesized in situ using light-directedmethods, flow channel and spotting methods, inkjet methods, pin-basedmethods and bead-based methods set forth in the following references:McGall et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:13555; SyntheticDNA Arrays In Genetic Engineering, Vol. 20:111, Plenum Press (1998);Duggan et al. (1999) Nat. Genet. S21:10; Microarrays: Making Them andUsing Them In Microarray Bioinformatics, Cambridge University Press,2003; U.S. Patent Application Publication Nos. 2003/0068633 and2002/0081582; U.S. Pat. Nos. 6,833,450, 6,830,890, 6,824,866, 6,800,439,6,375,903 and 5,700,637; and PCT Publication Nos. WO 04/031399, WO04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO 03/065038, WO03/064699, WO 03/064027, WO 03/064026, WO 03/046223, WO 03/040410 and WO02/24597; the disclosures of which are incorporated herein by referencein their entirety for all purposes. In some embodiments, pre-synthesizedoligonucleotides are attached to a support or are synthesized using aspotting methodology wherein monomers solutions are deposited dropwiseby a dispenser that moves from region to region (e.g., ink jet). In someembodiments, oligonucleotides are spotted on a support using, forexample, a mechanical wave actuated dispenser.

In some embodiments, each solid support comprises a fiber opticemitter/detector, therefore assessing the status of the reaction (e.g.assessing if the solid support is in the reagent when in an activatedstate). In certain embodiments, each feature or spot on the solidsupport can be assessed with a fiber optic emitter/detector to assessthe status of the reaction for each feature or spot. For example, thestatus (e.g. activated state or resting state) of each feature or spotcan be assessed to determine if the activated solid supports are incontact with the reagent and if the resting solid support are not incontact with the reagent. Similarly, the reaction status can be assessedby electrical measurement. In one embodiment, the solid supports aremade of conductive material and can serve as a probe to detect if thesolid supports are in contact with a liquid such as a reagent solution.A current flow can be induced in the reagent vessel and the current canthen be measured on the solid support side using, for example, anamperometer.

It should be appreciated that the device can include more than one solidsupports. In some embodiments, two or more solid supports can bearranged on a solid support holder, with each solid support beingmonitored by a same or a different emitter/detector. Any arrangement ofthe solid supports could be employed. In some embodiments, the solidsupports are arranged in rows and columns. The holder can comprise onerow or one column or a plurality of rows and columns. In someembodiments, the solid supports are arranged in rows and columns andeach row and column are equally spaced. For example, the rows can bealigned and/or the columns can be aligned. In other embodiments, rowsand/or columns are equally spaced and staggered. Spacing between rowsand/or between columns can be variable. In some embodiments, thedistance apart two adjacent solid supports is in the range of about 100μm to 5 mm, usually about 1 to 2 mm. The number of the solid supportscomprised in the holder can be variable. In an exemplary embodiment, theholder holds at least 10, at least 100, at least 500, at least 1000, atleast 2000 solid supports, at least 5,000, at least 10,000 or at least50,000 solid supports. In one embodiment, the holder holds 1536 solidsupports or pins.

In some embodiments, a vessel corresponding to each feature/spot or theentire solid support contains the reagents or the washing solution.Vessels can be adapted to the solid support dimension and shape. Eachstep in the synthesis can be carried simultaneously in the same vessel.In one embodiment where more than one solid support is used, at eachstep of the synthesis, all activated solid supports can be in fluidcommunication with each others. In other embodiments, the vessels can bemicrotiter plates and at each or some of the step of the synthesis, theactivated solid supports are not in fluid communication with eachothers. In some embodiments, the vessels can be chilled or heated. Oneshould appreciate that by making use of a plurality of the surfacesynthesis areas simultaneously, one can minimize the volume of thereagents in the vessels (for example the washing and coupling steps inoligonucleotide synthesis) and therefore minimize the cost of theoverall polynucleotide synthesis. The elution step can be performed in adifferent type of vessel. For example, the elution step can be carriedout either by batch elution (i.e. in a same vessel) or by eluting inindependent vessels (e.g., wells of a microtiter plate of the like).

Degenerate Binding Sequences

In various embodiments, within each feature on the solid support, eachof the plurality of oligonucleotides includes an identical predeterminedsubunit sequence of X nucleosides and a degenerate sequence of Ynucleosides. In general, a sequence is called degenerate if some of itspositions have several possible bases. Assuming Σ={T, C, A, G} is theDNA alphabet, a sequence (e.g. a primer) can be shown as S=x₁x₂ . . .x₁, where x_(i) ⊂Σ, x_(i)≠Ø and l is the length of S. For example, inthe primer P*={G} {G} {C,G} {A} {T,C,G} {A} the third position is C or Gand the fifth is C, T or G. The IUPAC illustration of P* will be GGSABA.The degeneracy of a sequence is the number of unique sequencecombinations it contains, which can be calculated as d(S)=Π^(l)_(i=1)|x_(i)|. For example, d(P*)=1×1×2×1×3×1=6. Degenerate primers areuseful for amplifying several related genomic or cDNA sequences, andhave been exploited in various applications such as amplifying DNAsequences of homologous genes or genes from a particular protein familyand analysis of species diversity. Generally, degenerate PCR can beuseful in identifying new members of a gene family or orthologous genesfrom different organisms. In some embodiments, PCR methods usingdegenerate primers to amplify unknown DNA sequences that are related toa known DNA sequence or to amplify a mixture of related sequences in onePCR reaction, can be used. Degenerate primers can be a mix of primerswith similar sequences.

In various embodiments, degenerate binding sequences can be used toimprove the tolerance of a chain extension reaction such that any givenprimer (or any single-stranded oligonucleotide with a free 3′-OH group)can bind to the degenerate binding sequence and allow extension off ofthe primer therefrom. The primer can have a specific sequence. Theprimer can also have any degree of degeneracy. For example, a specificprimer P (e.g., having a linker or adapter sequence) can be introducedto a first feature F₁ having a first predetermined subunit sequence X₁(e.g., a payload). The primer can anneal to the degenerate bindingsequence and extend therefrom using the first predetermined subunitsequence X₁ as template, thereby producing a first product having thesequence of P-X₁. The first product can be removed from the firstfeature F₁ and introduced to a second feature F₂ having a secondpredetermined subunit sequence X₂. At the second feature F₂, P-X₁ cananneal to the degenerate binding sequence and extend therefrom using thesecond predetermined subunit sequence X₂ as template, thereby producinga second product having the sequence of P-X₁-X₂. These steps can berepeated n times until the desired product P-X₁-X₂-X₃ . . . -X_(n) isproduced. The number n can be any integral, e.g., 1, 2, 3, 4, 5, 6, 7,9, 10, 15, 20, 25, 30, or more. In some embodiments, features F₁, F₂, .. . F_(n) have fixed positions on the solid support (e.g., a universalarray or omni chip) and a droplet containing the reaction reagents canbe moved around or advanced from one feature to another via automatedmeans. In other embodiments, features F₁, F₂, . . . F_(n) can berearranged (e.g., sequentially) such that all desired predeterminedsubunit sequences X₁, X₂, . . . X_(n) are grouped and manipulatedtogether.

The unwanted primer sequence can then be removed to produce X₁-X₂-X₃ . .. -X_(n) (e.g., by cleaving P off via the linker or adapter sequencetherein). Alternatively, a pair of primers that are specific to theX₁-X₂-X₃ . . . -X_(n) sequence can be used to specifically amplifyX₁-X₂-X₃ . . . -X_(n). It should be understood that amplification of anyintermediate product (e.g., X₁-X₂-X₃) can also be used to increase theamount of desired molecules at any step of such sequential synthesis.The amplified intermediate product (e.g., X₁-X₂-X₃) can then be subjectto the next extension reaction (e.g., to produce X₁-X₂-X₃-X₄). In someembodiments, before the next extension reaction, the amplifiedintermediate product (e.g., X₁-X₂-X₃) can be additionally subject to aselection process to select for the proper plus or minus strand (e.g.,by denaturing the double-stranded molecules and hybridizing to aselection array).

In an exemplary embodiment, a primer/primer binding site that contains abinding and/or cleavage site for a type IIs restriction endonuclease maybe used to remove the unwanted primer. The term “type-IIs restrictionendonuclease” refers to a restriction endonuclease having anon-palindromic recognition sequence and a cleavage site that occursoutside of the recognition site (e.g., from 0 to about 20 nucleotidesdistal to the recognition site). Type IIs restriction endonucleases maycreate a nick in a double-stranded nucleic acid molecule or may create adouble-stranded break that produces either blunt or sticky ends (e.g.,either 5′ or 3′ overhangs). Examples of Type IIs endonucleases include,for example, enzymes that produce a 3′ overhang, such as, for example,Bsr I, Bsm I, BstF5 I, BsrD I, Bts I, Mnl I, BciV I, Hph I, Mbo II, EciI, Acu I, Bpm I, Mme I, BsaX I, Bcg I, Bae I, Bfi I, TspDT I, TspGW I,Taq II, Eco57 I, Eco57M I, Gsu I, Ppi I, and Psr I; enzymes that producea 5′ overhang such as, for example, BsmA I, Ple I, Fau I, Sap I, BspM I,SfaN I, Hga I, Bvb I, Fok I, BceA I, BsmF I, Ksp632 I, Eco31 I, Esp3 I,Aar I; and enzymes that produce a blunt end, such as, for example, Mly Iand Btr I. Type-IIs endonucleases are commercially available and arewell known in the art (New England Biolabs, Beverly, Mass.).

In some embodiments, X is between 2 and 50 nucleosides. Moreparticularly, X is between 3 and 20. In some examples, X is 3, 4, 5, 6,7, 8, 9, or 10. In certain embodiments, Y is between 5 and 100. Moreparticularly, Y is between 5 and 20, or Y is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the device canhave at least 4^(X) different features. In some examples, the device hasat least 100, 1,000, 4,000, 10,000 or more different features. Thepredetermined subunit sequences can be different between the features. Asubset of the plurality of the features together can represent thepolynucleotide having the predetermined sequence of Z nucleosides. Incertain embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 98%, 99% or more of all possible combinations (4^(Z)) of thepolynucleotide having the predetermined sequence of Z nucleosides arerepresented on a single device.

In certain embodiments, the plurality of binding sequences aredegenerate sequences having a length of N1 nucleosides, and theplurality of binding sequences comprise up to 4^(N1) differentsequences. In some embodiments, N1 is between 5 and 100. In someembodiments, N1 is between 10 and 50. In some examples, N1 is 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one example, N1is 10. In some embodiments, the subunit sequence has a length of N2 andis one of 4^(N2) possible sequences. The solid support includes 4^(N2)spots or a subset or superset thereof. In some embodiments, N2 isbetween 2 and 50. In some embodiments, N2 is between 4 and 20. In someexamples, N2 is 3, 4, 5, 6, 7, 8, 9, or 10. In one example, N2 is 5.

With reference to FIG. 2A, an exemplary device having a feature (210)which comprises a plurality of oligonucleotides (201) having adegenerate binding sequence (203) and a payload sequence (202) isillustrated. The feature can contain multiple copies of oligonucleotides(e.g., 10³, 10⁴, 10⁵, 10⁶, 10⁷, etc.). A plurality of features exists ona solid support, and the number of features can be between, e.g., 1 to100,000,000. In this example, the payload sequence (202) (apredetermined subunit sequence) is 5 nucleoside long (AGTCA) and thedegenerate binding sequence (203) is 10 nucleosides long (NNNNNNNNNN).Therefore, to synthesize, e.g., a predetermined 60-mer starting withAGTCA, a total of 12 different features (each having a specific 5-merpayload sequence) can be selected, and an automated path starting fromthe feature having the AGTCA payload is determined. Then a dropletcontaining the reaction reagents can be moved around or advanced fromone feature to the next via automated means. The droplet, after runningthrough the 12 selected features (a run), contains the desired product(the predetermined 60-mer).

In some embodiments, to increase output, multiple runs through the samepredetermined path can be used to produce multiple copies of the desiredproduct. The runs can proceed consecutively, with a first run one ormore steps ahead of a second run. Multiple runs can also be manipulatedsimultaneously, e.g., on multiple solid supports in parallel.

Polynucleotide Synthesis/Assembly

In one aspect, the invention relates to a method for producing highfidelity polynucleotides on a solid support. The syntheticpolynucleotides are at least 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 40, 50,75, or 100 nucleosides in length, or longer. In exemplary embodiments, acomposition of synthetic polynucleotides contains at least about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 50%, 60%, 70%, 80%, 90%,95% or more copies that have a desired sequence that does not deviatefrom a predetermined sequence. In certain embodiments, at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more of allpossible combinations (4^(z)) of the polynucleotide having thepredetermined sequence of Z nucleosides are represented on a singledevice.

Some aspects the invention relate to the design of oligonucleotides forhigh fidelity polynucleotide assembly. Aspects of the invention may beuseful to increase the throughput rate of a nucleic acid assemblyprocedure and/or reduce the number of steps or amounts of reagent usedto generate a correctly assembled nucleic acid. In certain embodiments,aspects of the invention may be useful in the context of automatednucleic acid assembly to reduce the time, number of steps, amount ofreagents, and other factors required for the assembly of each correctnucleic acid. Accordingly, these and other aspects of the invention maybe useful to reduce the cost and time of one or more nucleic acidassembly procedures.

In some embodiments, the method includes synthesizing a plurality ofoligonucleotides or polynucleotides in a chain extension reaction usinga plurality of single stranded oligonucleotides as templates. As notedabove, the oligonucleotides may be first synthesized onto a plurality ofdiscrete features of the surface, or may be deposited on the pluralityof features of the support. The support may comprise at least 100, atleast 1,000, at least 10⁴, at least 10⁵, at least 10⁶, at least 10⁷, atleast 10⁸ features. In a preferred embodiment, the oligonucleotides arecovalently attached to the support. In preferred embodiments, the firstplurality of oligonucleotides is immobilized to a solid surface. In apreferred embodiment, each feature of the solid surface comprises a highdensity of oligonucleotides, each oligonucleotide having a differentpredetermined sequence (e.g., approximately 10⁶-10⁸ molecules perfeature).

In some embodiments, pluralities of different single-strandedoligonucleotides immobilized at different features of a solid support.In some embodiments the oligonucleotides may be attached through their5′ end. In a preferred embodiment, the oligonucleotides are attachedthrough their 3′ end. It should be appreciated that by 3′ end, it ismeant the sequence downstream to the 5′ end and by 5′ end it is meantthe sequence upstream to the 3′ end. For example, an oligonucleotide maybe immobilized on the support via a nucleotide sequence (e.g., adegenerate binding sequence), linker or spacer (e.g., a moiety that isnot involved in hybridization). In some embodiments, the first pluralityof oligonucleotides has a 3′ end that is complementary to the 3′ end ofan input single-stranded oligonucleotide. In some embodiments, if thetarget polynucleotide requires N extension cycles, 1 to N pluralities ofdifferent support-bound single stranded oligonucleotides are designedsuch that collectively the N oligonucleotide sequences comprise thetarget polynucleotide sequence.

It should be appreciated that different oligonucleotides may be designedto have different lengths. In some embodiments, one or more differentoligonucleotides may have overlapping sequence regions (e.g.,overlapping 5′ regions or overlapping 3′ regions). Overlapping sequenceregions may be identical (i.e., corresponding to the same strand of thenucleic acid fragment) or complementary (i.e., corresponding tocomplementary strands of the nucleic acid fragment). Overlappingsequences may be of any suitable length. Overlapping sequences may bebetween about 5 and about 500 nucleotides long (e.g., between about 10and 100, between about 10 and 75, between about 10 and 50, about 20,about 25, about 30, about 35, about 40, about 45, about 50, etc.)However, shorter, longer or intermediate overlapping lengths may beused. It should be appreciated that overlaps between different inputnucleic acids used in an assembly reaction may have different lengths.In some embodiments, immobilized oligonucleotides include sequenceregions having overlapping regions to assist in the assembly of apredetermined nucleic acid sequence. In a preferred embodiment,immobilized oligonucleotides include sequence regions havingcomplementary regions for hybridization to a different oligonucleotideor to a polynucleotide. The complementary regions refer to a sequenceregion at either a 3′ end or a 5′ end of the immobilized templateoligonucleotide. In a preferred embodiment, the complementary region islocalized at the 3′ end of the immobilized oligonucleotides.Complementary regions refer to a 3′ end or a 5′ region of a firstoligonucleotide or polynucleotide that is capable of hybridizing to a 5′end or 3′ end of a second oligonucleotide or polynucleotide.

FIG. 1 shows an exemplary method for producing polynucleotide on asubstrate or solid support. The method comprises several repeated stepsof annealing, extension and melting on different features (102, 103,104, 105) of the solid support (FIG. 1A-1F). With regards to FIG. 1,each feature of the solid support comprises a plurality of molecules(106) having a predefined sequence. In some embodiments, the pluralityof molecules having predefined sequences formed the final polynucleotideproducts. Yet in other embodiment, the plurality molecules having apredefined sequence partially comprise a sequence of the final product.

In some embodiments, a population of free (i.e., non-immobilized) inputpolynucleotides (element 101, FIG. 1A) is added to a first feature ofthe solid support (for example feature 102, FIG. 1A). In a preferredembodiment, the input polynucleotides are single strandedpolynucleotides (single stranded DNA for example). The inputpolynucleotide may be a synthetic oligonucleotide that is synthesized orobtained from a commercial supplier. In some embodiments, one or moreinput nucleic acids may be amplification products (e.g., PCR products),restriction fragments, or other suitable nucleic acid molecules. In someembodiments, the first plurality of oligonucleotides is designed to havea 3′ sequence that is complementary to the 3′ end of the inputpolynucleotide. Yet, in other embodiments, the input polynucleotidesequence of (101) is designed such that the 3′ a terminal sequence (107)hybridizes to a region (157) of the oligonucleotide sequence (106).

In a first step, the polynucleotide sequence (101) is partiallyhybridized to the support-bound oligonucleotide, the hybridized regionbeing formed between the (107) region of the polynucleotide and the(157) region of the immobilized oligonucleotide as shown in FIG. 1B. Ina second step, polymerase-mediated extension of the hybridizedpolynucleotides results in a template-based extension of the 3′ ends ofpolynucleotide nucleotides that have annealed 3′ regions generatingextended polynucleotides containing sequences that are complementary toa sequence region of the template oligonucleotide. Referring to FIG. 1C,the polynucleotide (101) is extended by addition of an appropriatepolymerase enzyme and other appropriate components (such as dNTPs, salt,buffer, and etc.) to allow the extension of polynucleotide (101) into alonger polynucleotide (110) that includes sequence (108) that iscomplementary to sequence (158) of the template oligonucleotide (106).The resulting molecule (110), now elongated by the length of sequence(158) is composed of the sequences of (101) and (108). In a third step,the extension product (110) is melted from oligonucleotide (106) andreleased into solution (FIG. 1D). The input polynucleotide can then betransferred to a different feature of the support. The inputpolynucleotide can be transferred or moved mechanically (e.g. pipetting)or using electric, electrostatic, electromagnetic forces. The inputpolynucleotide (110) can then partially anneal due to the complementarysequence region between polynucleotide (110) and oligonucleotide (111)immobilized on a different feature of the solid support (for example(111), feature (103) of the solid support (100), FIG. 1E)

The annealing of input polynucleotide (110) to oligonucleotide (111),followed by its extension as described above, leads to a longerpolynucleotide (120) comprising sequences of polynucleotide (101) andcomplementary sequences of oligonucleotides (106), (111) and (121). Withregards to FIG. 1, in region (103), the population of molecules(intended to be identical to 111) is designed with a sequence region(167) that hybridizes to sequence region (108) of polynucleotide (110),forming a hybridized region (112) composed of the sequences of (108) and(167). The addition of a polymerase with other appropriate components(such as dNTPs, salt, buffer, and etc.) allows for the extension ofpolynucleotide (110) to include sequence (113) using sequence region(168) as template. The resulting molecule (120), now elongated by thelength of sequence (168) and is composed of the sequences of (101),(108), and (113). The molecule can be melted from oligonucleotide (111)and released into solution, allowing it to hybridize to a differentregion (104) of the surface (100) (FIG. 1E). This process can berepeated to allow the elongation of (120) to include sequence region(123) from sequence region (104) and sequence region (133) from region(105), resulting in sequence (140) (FIG. 1F).

These cycles of melting, transferring, annealing and extension may berepeated until the target full length polynucleotide having apredetermined sequence is synthesized, each cycle of polymeraseextension extending oligonucleotide pairs with annealed 3′ regions. Ineach cycle, extension results in the addition of sequences complementaryto the template oligonucleotide. Each cycle may include a denaturing,transferring, annealing and extension step. However, the extension mayoccur under the annealing conditions. Accordingly, in one embodiment,cycles of extension may be obtained by alternating between denaturingconditions (e.g., a denaturing temperature) and annealing/extensionconditions (e.g., an annealing/extension temperature). However, in someembodiments, progressive extension may be achieved without temperaturecycling. For example, an enzyme capable of promoting rolling circleamplification may be used (e.g., TempliPhi). It should be appreciatedthat several cycles of polymerase extension may be required to assemblea single target polynucleotide containing the sequences of an initialplurality of template oligonucleotides. In some embodiments, the processcan be carried out for M steps, where M can be greater than 1, greaterthan 10, greater than 100, greater than 1,000, greater than 10,000,greater than 100,000. In some embodiments, the number of cycles is equalor superior to the number of immobilized oligonucleotides. A full lengthproduct (or predetermined target polynucleotide sequence) may beisolated or purified using a size selection, cloning, selective bindingor other suitable purification procedure. In addition, the full lengthproduct may be amplified using appropriate 5′ and 3′ amplificationprimers.

Polymerase-based assembly techniques may involve one or more suitablepolymerase enzymes that can catalyze a template-based extension of anucleic acid in a 5′ to 3′ direction in the presence of suitablenucleotides and an annealed template. A polymerase may be thermostable.A polymerase may be obtained from recombinant or natural sources. Insome embodiments, a thermostable polymerase from a thermophilic organismmay be used. In some embodiments, a polymerase may include a 3′→5′exonuclease/proofreading activity. In some embodiments, a polymerase mayhave no, or little, proofreading activity (e.g., a polymerase may be arecombinant variant of a natural polymerase that has been modified toreduce its proofreading activity). Examples of thermostable DNApolymerases include, but are not limited to: Taq (a heat-stable DNApolymerase from the bacterium Thermus aquaticus); Pfu (a thermophilicDNA polymerase with a 3′→5′ exonuclease/proofreading activity fromPyrococcus furiosus, available from for example Promega); VentR® DNAPolymerase and VentRO (exo-) DNA Polymerase (thermophilic DNApolymerases with or without a 3′→5′ exonuclease/proofreading activityfrom Thermococcus litoralis; also known as Th polymerase); Deep VentR®DNA Polymerase and Deep VentR® (exo-) DNA Polymerase (thermophilic DNApolymerases with or without a 3′→5′ exonuclease/proofreading activityfrom Pyrococcus species GB-D; available from New England Biolabs); KODHiFi (a recombinant Thermococcus kodakaraensis KODI DNA polymerase witha 3′→5′ exonuclease/proofreading activity, available from Novagen);BIO-X-ACT (a mix of polymerases that possesses 5′-3′ DNA polymeraseactivity and 3′→5′ proofreading activity); Klenow Fragment (anN-terminal truncation of E. coli DNA Polymerase I which retainspolymerase activity, but has lost the 5′ 3′ exonuclease activity,available from, for example, Promega and NEB); Sequenase™ (T7 DNApolymerase deficient in T-5′ exonuclease activity); Phi29 (bacteriophage29 DNA polymerase, may be used for rolling circle amplification, forexample, in a TempliPhi™ DNA Sequencing Template Amplification Kit,available from Amersham Biosciences); TopoTaq (a hybrid polymerase thatcombines hyperstable DNA binding domains and the DNA unlinking activityof Methanopyrus topoisomerase, with no exonuclease activity, availablefrom Fidelity Systems); TopoTaq HiFi which incorporates a proofreadingdomain with exonuclease activity; Phusion™ (a Pyrococcus-like enzymewith a processivity-enhancing domain, available from New EnglandBiolabs); any other suitable DNA polymerase, or any combination of twoor more thereof. In some embodiments, the polymerase can be a SDP(strand-displacing polymerase; e.g, an SDPe—which is an SDP with noexonuclease activity). This allows isothermal PCR (isothermal extension,isothermal amplification) at a uniform temperature. As the polymerase(for example, Phi29, Bst) travels along a template it displaces thecomplementary strand (e.g., created in previous extension reactions). Asthe displaced DNAs are single-stranded, primers can bind at a consistenttemperature, removing the need for any thermocycling duringamplification.

In some embodiments, the first step of the extension reaction uses aprimer (or seed primer). In some embodiments, the oligonucleotides maycomprise universal (common to all oligonucleotides), semi-universal(common to at least of portion of the oligonucleotides) or individual orunique primer (specific to each oligonucleotide) binding sites on eitherthe 5′ end or the 3′ end or both. As used herein, the term “universal”primer or primer binding site means that a sequence used to amplify theoligonucleotide is common to all oligonucleotides such that all sucholigonucleotides can be amplified using a single set of universalprimers. In other circumstances, an oligonucleotide contains a uniqueprimer binding site. As used herein, the term “unique primer bindingsite” refers to a set of primer recognition sequences that selectivelyamplifies a subset of oligonucleotides. In yet other circumstances, anoligonucleotide contains both universal and unique amplificationsequences, which can optionally be used sequentially. In a first step, aprimer is added and anneals to an immobilized oligonucleotide. In someembodiments, the support bound or immobilized oligonucleotides comprisea primer binding site wherein the primer is complementary to the primerbinding site. In the first step, a solution comprising a polymerase, atleast one primer and dNTPs is added at a feature of the solid supportunder conditions promoting primer extension.

One should appreciate that the extension reactions can take place in asingle volume that encompasses all of the utilized features (102, 103,104, 105, . . . ), or each step can take place in a localized individualvolume (e.g. droplet) that contains only the region(s) of interestduring a specific elongation step (see U.S. provisional application61/235,677, U.S. provisional application 61/257,591 filed on Nov. 3,2009, U.S. provisional applications 61/264,632 and 61/264,641, filed onNov. 25, 2009 and PCT application PCT/US09/55267, which are incorporateherein by reference in their entirety). In some embodiments, it may beimportant to control the seed primer (or the first input polynucleotide)concentration. When performing the extension reactions in a singlevolume (e.g. pooled extension), the extension product at step N, aftermelted off the surface-bond template, is free to hybridize to anysurface-bond extension template such as (N), (N-1), (N-2), . . . all theway down to the first extension template. Indeed, the extension producthaving complementary sequence to all of the “prior-step” templates willresult in side reactions (side hybridizations) and therefore will reducethe concentration of the polynucleotide of interest. In someembodiments, by increasing the concentration of the initial seed primerconcentration, it is possible to correct for the side reactions. In anexemplary embodiment, if the support-bound templates have on average Cnumber of oligonucleotides for each feature (i.e. each step of theextension), and M is the number of total extension steps, it is possibleto introduce C*M number of seed molecules at the first step to correctfor the side reactions.

In some embodiments, only a selected set of oligonucleotides suitablefor hydration are hydrated while the remainder of the support remainsdry. In one embodiment, each oligonucleotide has a predefined sequencedifferent from the predefined sequence of the oligonucleotide bound to adifferent feature. In some embodiments, a set of predefined features maybe selectively hydrated, thereby providing hydrated oligonucleotides. Inanother embodiment, the hydrated oligonucleotides are exposed to furtherprocessing within a droplet volume. For example, during the stepsillustrated by FIGS. 1A, 1B and 1C, only region (102) may be covered byan isolated liquid volume or droplet, the droplet acting as a virtualreaction chamber. The liquid volume (or aqueous phase) may comprisewater, buffer, primers, master mix, release chemicals, enzymes, or anycombination thereof. For example the solution may be composed ofoligonucleotides primer(s), nucleotides (dNTPs), buffer, polymerase andcofactors. In other embodiments, the solution is an alkaline denaturingsolution. Yet, in other embodiments, the solution may compriseoligonucleotides such as complementary oligonucleotides or inputpolynucleotide. After melting of the extension product (110), the liquidvolume or droplet is moved from region (102) to (103), carrying themelted extension products in solution to region (103). This process ofmoving the liquid volume can be repeated for each extension step of theprocess.

Other aspects of the invention relate to methods and devices forassembling at least one polynucleotide having a predefined sequence on asupport. In one embodiment, a support is provided that comprises atleast one feature having a plurality of surface-bound single-strandedoligonucleotides that are in a dry form and suitable for hydration. Eachplurality of oligonucleotides is bound to a discrete feature of thesupport, and the predefined sequence of each plurality ofoligonucleotides attached to the feature is different from thepredefined sequence of the plurality of oligonucleotides attached to adifferent feature. At least one feature is hydrated thereby providinghydrated oligonucleotides within a droplet. The support (or part of thesupport) is then heated to a first melting temperature under a firststringent melt condition thereby denaturing unstable duplexes (e.g., theduplexes not bound by a stabilizing agent). The melted, unextendedsingle-stranded molecules can be removed from the droplet (e.g., by awash buffer). With the stable duplexes (e.g., the duplexes bound by astabilizing agent), at least one plurality of oligonucleotides issynthesized in a chain extension reaction on a first feature of thesupport by template-dependent synthesis. The products of chain extensionare subjected to a second round of denaturation (e.g., at a secondmelting temperature) and the resulting extended, single-strandedoligonucleotides are removed from the support. Alternatively, themelted, unextended single-stranded molecules can remain within thedroplet after the first stringent melt; this way after the second roundof denaturation the resulting oligonucleotides can include bothunextended and extended molecules. This pool of unextended and extendedmolecules can be subjected to further selection (e.g., PCR,electrophoresis, etc.) to enrich for extended molecules. These steps canbe repeated on at least one other feature until the desired lengthand/or sequence is produced.

A first droplet comprising a first plurality of oligonucleotides canthen be combined to a second droplet comprising a second plurality ofoligonucleotides, wherein a terminal region of the second plurality ofoligonucleotides comprises identical or complementary sequences with aterminal region of the first set of plurality of oligonucleotides andthe first and second plurality of oligonucleotides are contacted underconditions that allow one or more of annealing, chain extension, anddenaturing. In some embodiments, the first and second droplets arecombined by merging the droplets into a second stage droplet. Firstand/or second droplets can be moved from a first feature to a secondfeature of the support. In some embodiments, the surface is coated witha low melting-point substance for storage, for example wax, for storage.In some embodiments, the reactions are initiated by heating the surfaceabove the low-melting point. Yet in other embodiments, the reactions areinitiated by hydrating the discrete features. In some embodiments, thesupport is a microfluidic device. Droplet movement may be controlled bythe flow rates of the fluid in the device or by electrical, magnetic,mechanical action applied to the droplets. The droplets and/or the fluidwithin the microfluidic device can be transported and distributed by avariety of forces including electric forces, electrokinetic forces,pressure based flow techniques, capillary forces, thermo-capillaryforces, gravitational and centrifugal forces, magnetic field, amechanical force, including mechanical pressure waves such as soundwaves or ultrasound, or an optical induced force or any combinationthereof. One should appreciate that isolated volumes may be routedindependently in a sequential or highly parallel fashion. Droplets maybe routed using electrowetting-based techniques (see for example, U.S.Pat. No. 6,911,132 and U.S. Patent Application 2006/0054503).Electrowetting principle is based on manipulating droplets on a surfacecomprising an array of electrodes and using voltage to change theinterfacial tension. In some embodiments, droplets are moved using awettability gradient. It has been shown that droplets placed onwettability gradient surfaces typically move in the direction ofincreasing wettability (see Zielke and Szymczyk, Eur. Phys. J. SpecialTopics, 166, 155-158 (2009)). In other embodiments, droplets may bemoved using a thermal gradient. When placed on a thermal gradient,droplets move from higher temperature locations towards lowertemperature locations. Moving droplets using electrowetting, temperaturegradients and wettability gradients depends on the liquid (e.g.,aqueous, non-aqueous, solute concentration), the size of the dropletsand/or the steepness of the gradient.

The manipulation of fluids to form fluid streams of desiredconfiguration, such as discontinuous fluid streams, particles,dispersions, etc., for purposes of fluid delivery, product manufacture,analysis, and the like, is a relatively well-studied art. See forexample, WO/2004/002627 which is incorporated herein in its entirety. Insome aspects of the invention, microfluidic devices are used to form andmanipulate droplets in a co-planar fashion to allow oligonucleotidesynthesis. For example, oligonucleotides may be synthesized using aphosphoramidite method. The phosphoramidite method, employingnucleotides modified with various protecting groups, is one of the mostcommonly used methods for the de novo synthesis of oligonucleotides.Detailed procedures for the phosphoramidite and hydrogen phosphonatemethods of oligonucleotide synthesis are described in the followingreferences that are incorporated by reference: U.S. Pat. Nos. 4,500,707;4,725,677; and 5,047,524. See also for example, methods outlined inOligonucleotide and Analogs: A practical approach, F. Eckstein, Ed. IRLPress Oxford University and Oligonucleotide synthesis: A practicalapproach, Gait, Ed. IRL Oxford Press. Synthesis can be performed eitherthrough the coupling of the 5′ position of the first monomer to the 3′position of the second monomer (3′-5′ synthesis) or vive versa (5′-3′synthesis). Briefly, synthesis of oligonucleotides requires the specificformation of a 3′-5′ or 5′-3′ phosphodiester linkage. In order to formthese specific linkages, the nucleophilic centers not involved in thelinkage must be chemically protected through the use of protectinggroup. By “protecting group” as used herein is meant a species whichprevents a segment of a molecule (e.g. nucleotide) from undergoing aspecific chemical reaction, but which is removable from the moleculefollowing completion of that reaction. For example, the 5′ hydroxylgroup may be protected by dimethoxitrityl (DMT). During the deblockingreaction, the DMT is removed with an acid, such as thrichloroacetic acid(TeA) or dichloroacetic acid, resulting in a free hydroxyl group. Afterwashing, a phosphoramidite nucleotide is activated by tetrazole,ethylthiotetrazole, dicyanoimidazole, or benzimidazolium triflate, forexample, which remove the iPr2N group on the phosphate group. Thedeprotected 5′ hydroxyl of the first base reacts with the phosphate ofthe second base and a 5′-3′ linkage is formed (coupling step). Unboundbases are washed out and 5′ hydroxyl group that did not react during thecoupling reaction are blocked by adding a capping group, whichpermanently binds to the free 5′ hydroxyl groups to prevent any furtherchemical transformation of that group (capping step). The oxidation stepmay be performed before or after the capping step. During oxidation, thephosphite linkage is stabilized to form a much more stable phosphatelinkage. The deblocking/coupling/capping/oxidation cycle may be repeatedthe requisite number of time to achieve the desired lengthpolynucleotide. In some embodiments, coupling can be synchronized on thearray or solid support.

In some embodiments, the oligonucleotides synthesis is synthesized usinga device that generates emulsion droplets comprising aqueous dropletswithin immiscible oil. The droplets may comprise an aqueous phase, animmiscible oil phase, and a surfactant and/or other stabilizingmolecules to maintain the integrity of the droplet. In some embodiments,mechanical energy is applied, allowing dispersion of a compound into anoil phase to form droplets, each of which contains a single sort ofcompound. Preferably, the compound is a nucleotide monomer (i.e. A, T orU, G, C). The compounds can be deposited into the oil phase in the formof droplets generated using inkjet printing technology or piezoelectricdrop-on-demand (DOD) inkjet printing technology. Each droplet maycomprise a different nucleotide monomer (i.e. A, T or U, G, C) in thesame aqueous solution. In preferred embodiments, the droplets areuniform in size and contain one nucleotide at a fixed concentration. Thedroplets can range in size from 0.5 microns to 500 micron in diameter,which correspond to a volume of about 1 picoliter to about 1 nanoliter.Yet in other embodiments, the droplet may comprise a 2-mer, a 3-mer, a4-mer, a 6-mer or a 7-mer oligonucleotide. In some embodiments, thedroplets are deposited onto a substrate such as a microsubstrate, amicroarray or a microchip. The terms microsubstrate, microarray andmicrochip are used interchangeably herein. The droplets may be depositedusing a microfluidic nozzle. In some embodiments, the substrate may besubjected to wash, deblocking solution, coupling, capping and oxidationreactions to elongate the oligonucleotide.

In some embodiments, the droplets carrying the nucleotides can be movedusing electrowetting technologies. Electrowetting involves modifying thesurface tension of liquids on a solid surface using a voltage. Uponapplication of an electric field (e.g. alternating or direct), thecontact angle between the fluid and surfaces can be modified. Forexample, by applying a voltage, the wetting properties of a hydrophobicsurface can become increasingly hydrophilic and therefore wettable.Electrowetting principle is based on manipulating droplets on a surfacecomprising an array of electrodes and using voltage to change theinterfacial tension. In some embodiments, the array of electrode is notin direct contact with the fluid. In some embodiments, the array ofelectrode is configured such as the support has a hydrophilic side and ahydrophobic side. The droplets subjected to the voltage will movetowards the hydrophilic side. In some embodiments, the array or patternof electrodes is a high density pattern. One should appreciate that tobe used in conjunction with the phosphoramidite chemistry, the array ofelectrodes should be able to move droplets volumes ranging from 1 pL(and less) to 10 pL. Accordingly, aspects of the invention relate tohigh voltage complementary semi-conductor microfluidic controller. Insome embodiments, the high voltage complementary semi-conductor device(HV-CMOS) has an integrated circuit with high density electrode patternand high voltage electronics. In some embodiments, the voltage appliedis between 15V and 30V.

In some embodiments, the entire support or array containing the discretefeatures is subjected to thermocycling, annealing temperatureconditions, stringent melt temperature conditions, or denaturingtemperature conditions. Heating and cooling the support can be performedin any thermal cycle instrument. In other embodiments, one or morediscrete features are subjected to specific temperature conditions(annealing, extension, wash or melt). Thermocycling of selectedindependent features (being separated from each others) can be performedby locally heating at least one discrete feature. Discrete features maybe locally heated by any means known in the art. For example, thediscrete features may be locally heated using a laser source of energythat can be controlled in a precise x-y dimension thereby individuallymodulating the temperature of a droplet. In another example, thecombination of a broader beam laser with a mask can be used to irradiatespecific features. In some embodiments, methods to control temperatureon the support so that enzymatic reactions can take place on a support(PCR, ligation or any other temperature sensitive reaction) areprovided. In some embodiments, a scanning laser is used to control thethermocycling on distinct features on the solid support. The wavelengthused can be chosen from a wide spectrum (100 nm to 100,000 nm, i.e.,from ultraviolet to infrared). In some embodiments, the feature on whichthe droplet is spotted comprises an optical absorber or indicator. Insome other embodiments, an optical absorbent material can be added onthe surface of the droplet. In some embodiments, the solid support iscooled by circulation of air or fluid. The energy to be deposited can becalculated based on the absorbance behavior. In some embodiments, thetemperature of the droplet can be modeled using thermodynamics. Thetemperature can be measured by an LCD like material or any other in-situtechnology. Yet in another embodiment, the whole support can be heatedand cooled down to allow enzymatic reactions or other temperaturesensitive reactions to take place. One method to control the temperatureof the surface droplets is by using a scanning optical energy depositionsetup. An energy source can be directed by a scanning setup to depositenergy at various locations on the surface of the solid supportcomprising support-bound molecules. Optical absorbent material can beadded on the surface of the solid support or on the surface of droplet.Optical energy source, such as a high intensity lamp, laser, or otherelectromagnetic energy source (including microwave) can be used. Thetemperature of the different reaction sites can be controlledindependently by controlling the energy deposited at each of thefeatures.

In some embodiments, after extension or amplification, the polymerasemay be deactivated to prevent interference with the subsequent steps. Aheating step (e.g., high temperature) can denature and deactivate mostenzymes which are not thermally stable. Enzymes may be deactivated inpresence (e.g., within the droplet) or in the absence of liquid (e.g.,dry array). Heat deactivation on a dry support has the advantage todeactivate the enzymes without any detrimental effect on theoligonucleotides. In some embodiments, a non-thermal stable version ofthe thermally stable PCR DNA Polymerase may be used, although the enzymeis less optimized for error rate and speed. Alternatively, Epoxy dATPcan be use to inactivate the enzyme.

It should be appreciated that the description of the synthesis/assemblyreactions in the context of oligonucleotides is not intended to belimiting. For example, other polynucleotides (e.g., single-stranded,double-stranded polynucleotides, restriction fragments, amplificationproducts, naturally occurring polynucleotides, etc.) may be included inan assembly reaction, along with one or more oligonucleotides, in orderto generate a polynucleotide of interest.

Stringent Melt

As used herein the term “stringent” or “stringency” is used in referenceto the conditions of temperature, ionic strength, and the presence ofother compounds such as organic solvents, under which nucleic acidhybridizations are conducted. Hybridization stringency increases withtemperature and/or chemical properties such as the amounts of saltsand/or formamide in the hybridization solution during a hybridizationprocess. With “high stringency” conditions, nucleic acid base pairingwill occur only between nucleic acid fragments that have a highfrequency of complementary base sequences. Stringent conditions may beselected to be about 5° C. lower than the thermal melting point (Tm) fora given polynucleotide duplex at a defined ionic strength and pH. Thelength of the complementary polynucleotide strands and their GC contentwill determine the Tm of the duplex, and thus the hybridizationconditions necessary for obtaining a desired specificity ofhybridization. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of a polynucleotide sequence hybridizes to aperfectly matched complementary strand. In certain cases it may bedesirable to increase the stringency of the hybridization conditions tobe about equal to the Tm for a particular duplex. Appropriate stringencyconditions are known to those skilled in the art or may be determinedexperimentally by the skilled artisan. See, for example, CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-12.3.6; Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, N.Y; S. Agrawal (ed.) Methods inMolecular Biology, volume 20; Tijssen (1993) Laboratory Techniques inbiochemistry and molecular biology-hybridization with nucleic acidprobes, e.g., part I chapter 2 “Overview of principles of hybridizationand the strategy of nucleic acid probe assays”, Elsevier, N.Y.

Aspects of the invention relate to enhancing nucleic acidsynthesis/assembly procedures by using a stringent melt/wash step afterannealing of the polynucleotide to the immobilized oligonucleotidesthrough the complementary regions and prior to polymerase extension.Accordingly, aspects of the invention may be useful for increasing thefidelity of a nucleic acid synthesis/assembly reaction (e.g., increasingthe proportion of synthesized/assembled nucleic acids that have adesired predetermined polynucleotide or target sequence). In someaspects of the invention, the immobilized oligonucleotides comprise atleast two, at least three different and contiguous sequence regions. Asillustrated herewith, the stringent melt/wash step allows for thereduction of the extension of unstable duplexes. In some embodiments,the precise, controlled extension that results in thedesired/predetermined sequence relies on the difference in meltingtemperature between stable (or substantially stable) and unstable (orsubstantially unstable) duplexes. The use of a stringent melt/washconditions with precisely controlled temperature, allows for theunstably duplexed molecules to be washed away and removed from thereaction sites, achieving the overall goal of high-precision synthesis.

The conditions for stringent melt (e.g., a precise melting temperature)can be determined by observing a real-time melt curve. In an exemplarymelt curve analysis, PCR products are slowly heated in the presence ofdouble-stranded DNA (dsDNA) specific fluorescent dyes (e.g., SYBR Green,LCGreen, SYTO9 or EvaGreen). With increasing temperature the dsDNAdenatures (melts), releasing the fluorescent dye with a resultantdecrease in the fluorescent signal. The temperature at which the dsDNAmelts is determined by factors such as nucleotide sequence, DNA lengthand GC/AT ratio. Typically, G-C base pairs in a duplex are estimated tocontribute about 3° C. to the Tm, while A-T base pairs are estimated tocontribute about 2° C., up to a theoretical maximum of about 80-100° C.However, more sophisticated models of Tm are available and may be inwhich G-C stacking interactions, solvent effects, the desired assaytemperature and the like are taken into account. Melt curve analysis candetect a single base difference. Methods for accurate temperaturecontrol at individual features can be used as disclosed in U.S.Provisional application 61/264,591). In some embodiments, a stringentwash step with a carefully controlled temperature can melt and removethe error-containing input polynucleotides after annealing.

In some embodiments, during the stringent melt/wash step, it isdesirable to have a global stringent melt/wash temperature such thatstringent melt/wash can be achieved for all of the participatingfeatures under the same temperature condition. Accordingly, some aspectsof the invention relate to the design of oligonucleotides such as thestringent melt/wash temperature is the same or within a narrowtemperature window. For example, the pluralities of oligonucleotides aredesigned to have a melting temperature that is within 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20° C. In some embodiments, the length of thehybridization step is varied at different features. One shouldappreciate that by adjusting the hybridization step length, the meltingtemperature of each of the extension step can be controlled.

In some embodiments, each support-bound oligonucleotide involved in theextension reactions is designed to have each feature's meltingtemperature (individual stringent melt temperature) tuned towards thesame target melting temperature (global stringent melt temperature). Theindividual stringent melt temperatures can be tuned as close to theglobal target as it is possible by increasing or decreasing the lengthsof the individual junction QC sections. In some cases, it may not bepossible to design oligonucleotides which individual stringent melttemperatures is the same as the global stringent melt temperature. Insome embodiments, the support-bound oligonucleotides are designed tohave an individual melt temperature to be within a defined range to theglobal stringent melt temperature. In some embodiments, the definedtemperature range can be expressed as a temperature deviation from thetarget global stringent melt temperature, and can be of 0.001, 0.01,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1° C., or less than 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 40° C.

In some embodiments, the stringent melt condition includes controlling atemperature of the droplet at each of the plurality of features/spots onthe solid support. The temperature can be controlled at above apredetermined temperature which corresponds to an average annealingtemperature of the plurality of binding sequences. In some embodiments,the temperature is controlled at up to 20° C. above the predeterminedtemperature. In some examples, the temperature is controlled at 1, 2, 3,4, 5, 6, 7, 8, 9, or 10° C. above the predetermined temperature. Incertain examples, the temperature is controlled at above thepredetermined temperature such that duplexes stabilized by a bindingagent remain substantially annealed at said temperature. In one example,the binding agent is a polymerase or a subunit thereof.

FIG. 2B illustrates under an exemplary stringent melt condition (e.g., apredetermined temperature), stabilized duplex (220) proceeds to chainextension while non-stabilized duplex (240) does not. The featurecontains multiple copies of oligonucleotides having a degenerate bindingsequence 203 and supporting a plurality of oligonucleotides. It shouldbe understood that a plurality of features exist on a single solidsupport, and the number of features can be, e.g., between 1 to100,000,000. By way of example, FIG. 2B shows two support-boundoligonucleotides (220, 240) attached within the same feature on a solidsupport. A primer (or any single-stranded oligonucleotide with a free3′-OH group) in this example has two regions (223, 224) where region 223(having a specific sequence or any degree of degeneracy) binds to thedegenerate binding sequence 203 and allow extension off of the primertherefrom to produce region 222. This is possible because under thestringent melt condition (e.g., the predetermined temperature), thoseduplexes that can undergo extension reaction (e.g., bound by polymerase)are stable and remain annealed (stabilized duplex 220). On the otherhand, the non-stabilized duplexes 240 are only partially annealed to thedegenerate binding sequence, thus having an overhang 245 which is notbound by polymerase because its 3′-OH is not available for chainextension. Therefore, non-stabilized duplex 240 is not extended can beremoved from the droplet before proceeding to a next feature for furtherextension.

Automation

It can be advantageous that most of the manipulations be assisted orautomated. Such manipulation includes positioning solid support holdersor positioning solid support, controlling the temperature of thedroplets or vessels containing the reagents, rotating the platforms,controlling the fluid or drain systems, and the like. For example, thepositioning of the holder can be suitably controlled by a positionersuch as a computer controlled mechanical arm. Alternatively, the holderscan be held stationary and the droplets or vessels containing thereagents can be moved by appropriate controllers. Automatic holder orsolid support can be connected to, via electronic leads to an electroniccontrol unit. The polynucleotide synthesis device can also include arouter for internet connection, a personal computer. The personalcompute can control the high level operation of the device through aninterface. This operation can comprise the submitting of synthesis jobsand the monitoring of the sequence progression, as well as detecting anyfault conditions.

Aspects of the methods and devices provided herein may includeautomating one or more acts described herein. In some embodiments, oneor more steps of an amplification and/or assembly reaction may beautomated using one or more automated sample handling devices (e.g., oneor more automated liquid or fluid handling devices). Automated devicesand procedures may be used to deliver reaction reagents, including oneor more of the following: starting nucleic acids, buffers, enzymes(e.g., one or more ligases and/or polymerases), nucleotides, salts, andany other suitable agents such as stabilizing agents. Automated devicesand procedures also may be used to control the reaction conditions. Forexample, an automated thermal cycler may be used to control reactiontemperatures and any temperature cycles that may be used. In someembodiments, a scanning laser may be automated to provide one or morereaction temperatures or temperature cycles suitable for incubatingpolynucleotides. Similarly, subsequent analysis of assembledpolynucleotide products may be automated. For example, sequencing may beautomated using a sequencing device and automated sequencing protocols.Additional steps (e.g., amplification, cloning, etc.) also may beautomated using one or more appropriate devices and related protocols.It should be appreciated that one or more of the device or devicecomponents described herein may be combined in a system (e.g., a roboticsystem) or in a micro-environment (e.g., a micro-fluidic reactionchamber). Assembly reaction mixtures (e.g., liquid reaction samples) maybe transferred from one component of the system to another usingautomated devices and procedures (e.g., robotic manipulation and/ortransfer of samples and/or sample containers, including automatedpipetting devices, micro-systems, etc.). The system and any componentsthereof may be controlled by a control system.

Accordingly, method steps and/or aspects of the devices provided hereinmay be automated using, for example, a computer system (e.g., a computercontrolled system). A computer system on which aspects of the technologyprovided herein can be implemented may include a computer for any typeof processing (e.g., sequence analysis and/or automated device controlas described herein). However, it should be appreciated that certainprocessing steps may be provided by one or more of the automated devicesthat are part of the assembly system. In some embodiments, a computersystem may include two or more computers. For example, one computer maybe coupled, via a network, to a second computer. One computer mayperform sequence analysis. The second computer may control one or moreof the automated synthesis and assembly devices in the system. In otheraspects, additional computers may be included in the network to controlone or more of the analysis or processing acts. Each computer mayinclude a memory and processor. The computers can take any form, as theaspects of the technology provided herein are not limited to beingimplemented on any particular computer platform. Similarly, the networkcan take any form, including a private network or a public network(e.g., the Internet). Display devices can be associated with one or moreof the devices and computers. Alternatively, or in addition, a displaydevice may be located at a remote site and connected for displaying theoutput of an analysis in accordance with the technology provided herein.Connections between the different components of the system may be viawire, optical fiber, wireless transmission, satellite transmission, anyother suitable transmission, or any combination of two or more of theabove.

Each of the different aspects, embodiments, or acts of the technologyprovided herein can be independently automated and implemented in any ofnumerous ways. For example, each aspect, embodiment, or act can beindependently implemented using hardware, software or a combinationthereof. When implemented in software, the software code can be executedon any suitable processor or collection of processors, whether providedin a single computer or distributed among multiple computers. It shouldbe appreciated that any component or collection of components thatperform the functions described above can be generically considered asone or more controllers that control the above-discussed functions. Theone or more controllers can be implemented in numerous ways, such aswith dedicated hardware, or with general purpose hardware (e.g., one ormore processors) that is programmed using microcode or software toperform the functions recited above.

In this respect, it should be appreciated that one implementation of theembodiments of the technology provided herein comprises at least onecomputer-readable medium (e.g., a computer memory, a floppy disk, acompact disk, a tape, etc.) encoded with a computer program (i.e., aplurality of instructions), which, when executed on a processor,performs one or more of the above-discussed functions of the technologyprovided herein. The computer-readable medium can be transportable suchthat the program stored thereon can be loaded onto any computer systemresource to implement one or more functions of the technology providedherein. In addition, it should be appreciated that the reference to acomputer program which, when executed, performs the above-discussedfunctions, is not limited to an application program running on a hostcomputer. Rather, the term computer program is used herein in a genericsense to reference any type of computer code (e.g., software ormicrocode) that can be employed to program a processor to implement theabove-discussed aspects of the technology provided herein.

It should be appreciated that in accordance with several embodiments ofthe technology provided herein wherein processes are stored in acomputer readable medium, the computer implemented processes may, duringthe course of their execution, receive input manually (e.g., from auser).

Accordingly, overall system-level control of the synthesis/assemblydevices or components described herein may be performed by a systemcontroller which may provide control signals to the associated nucleicacid synthesizers, liquid handling devices, thermal cyclers, sequencingdevices, associated robotic components, as well as other suitablesystems for performing the desired input/output or other controlfunctions. Thus, the system controller along with any device controllerstogether form a controller that controls the operation of a nucleic acidassembly system. The controller may include a general purpose dataprocessing system, which can be a general purpose computer, or networkof general purpose computers, and other associated devices, includingcommunications devices, modems, and/or other circuitry or components toperform the desired input/output or other functions. The controller canalso be implemented, at least in part, as a single special purposeintegrated circuit (e.g., ASIC) or an array of ASICs, each having a mainor central processor section for overall, system-level control, andseparate sections dedicated to performing various different specificcomputations, functions and other processes under the control of thecentral processor section. The controller can also be implemented usinga plurality of separate dedicated programmable integrated or otherelectronic circuits or devices, e.g., hard wired electronic or logiccircuits such as discrete element circuits or programmable logicdevices. The controller can also include any other components ordevices, such as user input/output devices (monitors, displays,printers, a keyboard, a user pointing device, touch screen, or otheruser interface, etc.), data storage devices, drive motors, linkages,valve controllers, robotic devices, vacuum and other pumps, pressuresensors, detectors, power supplies, pulse sources, communication devicesor other electronic circuitry or components, and so on. The controlleralso may control operation of other portions of a system, such asautomated client order processing, quality control, packaging, shipping,billing, etc., to perform other suitable functions known in the art butnot described in detail herein.

Various aspects of the present invention may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

Application:

Aspects of the invention may be useful for a range of applicationsinvolving the production and/or use of synthetic nucleic acids. Asdescribed herein, the invention provides methods for producing syntheticnucleic acids with increased fidelity and/or for reducing the costand/or time of synthetic assembly reactions. The resulting assemblednucleic acids may be amplified in vitro (e.g., using PCR, LCR, or anysuitable amplification technique), amplified in vivo (e.g., via cloninginto a suitable vector), isolated and/or purified. An assembled nucleicacid (alone or cloned into a vector) may be transformed into a host cell(e.g., a prokaryotic, eukaryotic, insect, mammalian, or other hostcell). In some embodiments, the host cell may be used to propagate thenucleic acid. In certain embodiments, the nucleic acid may be integratedinto the genome of the host cell. In some embodiments, the nucleic acidmay replace a corresponding nucleic acid region on the genome of thecell (e.g., via homologous recombination). Accordingly, nucleic acidsmay be used to produce recombinant organisms. In some embodiments, atarget nucleic acid may be an entire genome or large fragments of agenome that are used to replace all or part of the genome of a hostorganism. Recombinant organisms also may be used for a variety ofresearch, industrial, agricultural, and/or medical applications.

In some embodiments, methods described herein may be used during theassembly of large nucleic acid molecules (for example, larger than 5,000nucleotides in length, e.g., longer than about 10,000, longer than about25,000, longer than about 50,000, longer than about 75,000, longer thanabout 100,000 nucleotides, etc.). In an exemplary embodiment, methodsdescribed herein may be used during the assembly of an entire genome (ora large fragment thereof, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or more) of an organism (e.g., of a viral, bacterial, yeast,or other prokaryotic or eukaryotic organism), optionally incorporatingspecific modifications into the sequence at one or more desiredlocations.

In one embodiment, the invention provides devices and methods forsynthesizing a plurality of polynucleotides. For example, the inventionprovides devices and methods for synthesizing a library ofpolynucleotides. In an exemplary embodiment, library polynucleotides areassembled from chemically synthesized oligonucleotides using techniquessuch as those set forth herein. In some embodiments, methods ofsynthesizing libraries containing nucleic acids having predeterminedsequence variations are provided herein. In some embodiments, librariesof nucleic acids are libraries of sequence variants. Sequence variantscan be variants of a single naturally-occurring protein encodingsequence. However, in some embodiments, sequence variants can bevariants of a plurality of different protein-encoding sequences.

Preferably, the polynucleotides are assembled in parallel from thechemically synthesized oligonucleotides. For example, in one embodiment,libraries can be constructed by hybridization based oligonucleotideassembly of overlapping complementary oligonucleotides (see e.g., Zhouet al., Nucleic Acids Res., 32: 5409-5417 (2004); Richmond et al.,Nucleic Acids Res. 32: 5011-5018 (2004); Tian et al. Nature 432:1050-1054 (2004); and Can et al. Nucleic Acids Res. 32: e162 (2004)).For example, oligonucleotides having complementary, overlappingsequences can be synthesized in parallel on a solid supports asdescribed herein and then eluted off. The oligonucleotides then selfassemble based on hybridization of the complementary regions. Thistechnique permits the production of long molecules of DNA having highfidelity and/or high precision sequences.

In some embodiments, the sequence of the polynucleotide construct can bedivided up into a plurality of shorter sequences that can be synthesizedin parallel and assembled into a single or a plurality of desiredpolynucleotide constructs using the methods described herein. In certainembodiments, the oligonucleotides are designed to provide the full senseand antisense strands of the polynucleotide construct. Afterhybridization of the plus and minus strand oligonucleotides, twodouble-stranded oligonucleotides are subjected to ligation in order toform a first subassembly product. Subassembly products are thensubjected to ligation to form a larger DNA or the full DNA sequence.

Ligase-based assembly techniques can involve one or more suitable ligaseenzymes that can catalyze the covalent linking of adjacent 3′ and 5′nucleic acid termini (e.g., a 5′ phosphate and a 3′ hydroxyl of nucleicacid(s) annealed on a complementary template nucleic acid such that the3′ terminus is immediately adjacent to the 5′ terminus). Accordingly, aligase can catalyze a ligation reaction between the 5′ phosphate of afirst nucleic acid to the 3′ hydroxyl of a second nucleic acid if thefirst and second nucleic acids are annealed next to each other on atemplate nucleic acid). A ligase can be obtained from recombinant ornatural sources. A ligase can be a heat-stable ligase. In someembodiments, a thermostable ligase from a thermophilic organism can beused. Examples of thermostable DNA ligases include, but are not limitedto: Tth DNA ligase (from Thermus thermophilus, available from, forexample, Eurogentec and GeneCraft); Pfu DNA ligase (a hyperthermophilicligase from Pyrococcus furiosus); Taq ligase (from Thermus aquaticus),any other suitable heat-stable ligase, or any combination thereof. Insome embodiments, one or more lower temperature ligases can be used(e.g., T4 DNA ligase). A lower temperature ligase can be useful forshorter overhangs (e.g., about 3, about 4, about 5, or about 6 baseoverhangs) that can not be stable at higher temperatures.

Non-enzymatic techniques can be used to ligate nucleic acids. Forexample, a 5′-end (e.g., the 5′ phosphate group) and a 3′-end (e.g., the3′ hydroxyl) of one or more nucleic acids can be covalently linkedtogether without using enzymes (e.g., without using a ligase). In someembodiments, non-enzymatic techniques can offer certain advantages overenzyme-based ligations. For example, non-enzymatic techniques can have ahigh tolerance of non-natural nucleotide analogues in nucleic acidsubstrates, can be used to ligate short nucleic acid substrates, can beused to ligate RNA substrates, and/or can be cheaper and/or more suitedto certain automated (e.g., high throughput) applications.

Non-enzymatic ligation can involve a chemical ligation. In someembodiments, nucleic acid termini of two or more different nucleic acidscan be chemically ligated. In some embodiments, nucleic acid termini ofa single nucleic acid can be chemically ligated (e.g., to circularizethe nucleic acid). It should be appreciated that both strands at a firstdouble-stranded nucleic acid terminus can be chemically ligated to bothstrands at a second double-stranded nucleic acid terminus. However, insome embodiments, only one strand of a first nucleic acid terminus canbe chemically ligated to a single strand of a second nucleic acidterminus. For example, the 5′ end of one strand of a first nucleic acidterminus can be ligated to the 3′ end of one strand of a second nucleicacid terminus without the ends of the complementary strands beingchemically ligated.

Accordingly, a chemical ligation can be used to form a covalent linkagebetween a 5′ terminus of a first nucleic acid end and a 3′ terminus of asecond nucleic acid end, wherein the first and second nucleic acid endscan be ends of a single nucleic acid or ends of separate nucleic acids.In one aspect, chemical ligation can involve at least one nucleic acidsubstrate having a modified end (e.g., a modified 5′ and/or 3′ terminus)including one or more chemically reactive moieties that facilitate orpromote linkage formation. In some embodiments, chemical ligation occurswhen one or more nucleic acid termini are brought together in closeproximity (e.g., when the termini are brought together due to annealingbetween complementary nucleic acid sequences). Accordingly, annealingbetween complementary 3′ or 5′ overhangs (e.g., overhangs generated byrestriction enzyme cleavage of a double-stranded nucleic acid) orbetween any combination of complementary nucleic acids that results in a3′ terminus being brought into close proximity with a 5′ terminus (e.g.,the 3′ and 5′ termini are adjacent to each other when the nucleic acidsare annealed to a complementary template nucleic acid) can promote atemplate-directed chemical ligation. Examples of chemical reactions caninclude, but are not limited to, condensation, reduction, and/orphoto-chemical ligation reactions. It should be appreciated that, insome embodiments, chemical ligation can be used to produce naturallyoccurring phosphodiester internucleotide linkages,non-naturally-occurring phosphamide pyrophosphate internucleotidelinkages, and/or other non-naturally-occurring internucleotide linkages.

In some embodiments, the process of chemical ligation can involve one ormore coupling agents to catalyze the ligation reaction. A coupling agentcan promote a ligation reaction between reactive groups in adjacentnucleic acids (e.g., between a 5′-reactive moiety and a 3′-reactivemoiety at adjacent sites along a complementary template). In someembodiments, a coupling agent can be a reducing reagent (e.g.,ferricyanide), a condensing reagent such (e.g., cyanoimidazole, cyanogenbromide, carbodiimide, etc.), or irradiation (e.g., UV irradiation forphoto-ligation).

In some embodiments, a chemical ligation can be an autoligation reactionthat does not involve a separate coupling agent. In autoligation, thepresence of a reactive group on one or more nucleic acids can besufficient to catalyze a chemical ligation between nucleic acid terminiwithout the addition of a coupling agent (see, for example, Xu et al.,(1997) Tetrahedron Lett. 38:5595-8). Non-limiting examples of thesereagent-free ligation reactions can involve nucleophilic displacementsof sulfur on bromoacetyl, tosyl, or iodo-nucleoside groups (see, forexample, Xu et al., (2001) Nat. Biotech. 19:148-52). Nucleic acidscontaining reactive groups suitable for autoligation can be prepareddirectly on automated synthesizers (see, for example, Xu et al., (1999)Nuc. Acids Res. 27:875-81). In some embodiments, a phosphorothioate at a3′ terminus can react with a leaving group (such as tosylate or iodide)on a thymidine at an adjacent 5′ terminus. In some embodiments, twonucleic acid strands bound at adjacent sites on a complementary targetstrand can undergo auto-ligation by displacement of a 5′-end iodidemoiety (or tosylate) with a 3′-end sulfur moiety. Accordingly, in someembodiments, the product of an autoligation can include anon-naturally-occurring internucleotide linkage (e.g., a single oxygenatom can be replaced with a sulfur atom in the ligated product).

In some embodiments, a synthetic nucleic acid duplex can be assembledvia chemical ligation in a one step reaction involving simultaneouschemical ligation of nucleic acids on both strands of the duplex. Forexample, a mixture of 5′-phosphorylated oligonucleotides correspondingto both strands of a target nucleic acid can be chemically ligated by a)exposure to heat (e.g., to 97° C.) and slow cooling to form a complex ofannealed oligonucleotides, and b) exposure to cyanogen bromide or anyother suitable coupling agent under conditions sufficient to chemicallyligate adjacent 3′ and 5′ ends in the nucleic acid complex.

In some embodiments, a synthetic nucleic acid duplex can be assembledvia chemical ligation in a two step reaction involving separate chemicalligations for the complementary strands of the duplex. For example, eachstrand of a target nucleic acid can be ligated in a separate reactioncontaining phosphorylated oligonucleotides corresponding to the strandthat is to be ligated and non-phosphorylated oligonucleotidescorresponding to the complementary strand. The non-phosphorylatedoligonucleotides can serve as a template for the phosphorylatedoligonucleotides during a chemical ligation (e.g., using cyanogenbromide). The resulting single-stranded ligated nucleic acid can bepurified and annealed to a complementary ligated single-stranded nucleicacid to form the target duplex nucleic acid (see, for example, Shabarovaet al., (1991) Nucl. Acids Res. 19:4247-51).

In one aspect, a nucleic acid fragment can be assembled in apolynucleotides-mediated assembly reaction from a plurality ofoligonucleotides that are combined and extended in one or more rounds ofpolynucleotides-mediated extensions. In some embodiments, theoligonucleotides are overlapping oligonucleotides covering the fullsequence but leaving single stranded gaps that can be filed in by chainextension. The plurality of different oligonucleotides can provideeither positive sequences, negative sequences, or a combination of bothpositive and negative sequences (e.g. plus and minus strands)corresponding to the entire sequence of the nucleic acid fragment to beassembled. In some embodiments, one or more different oligonucleotidescan have overlapping sequence regions (e.g., overlapping 5′ regions oroverlapping 3′ regions). Overlapping sequence regions can be identical(i.e., corresponding to the same strand of the nucleic acid fragment) orcomplementary (i.e., corresponding to complementary strands of thenucleic acid fragment). The plurality of oligonucleotides can includeone or more oligonucleotide pairs with overlapping identical sequenceregions, one or more oligonucleotide pairs with overlappingcomplementary sequence regions, or a combination thereof. Overlappingsequences can be of any suitable length. For example, overlappingsequences can encompass the entire length of one or more nucleic acidsused in an assembly reaction. Overlapping sequences can be between about5 and about 500 ucleotides long (e.g., between about 10 and 100, betweenabout 10 and 75, between about 10 and 50, about 20, about 25, about 30,about 35, about 45, about 50, etc.). However, shorter, longer, orintermediate overlapping lengths can be used. It should be appreciatedthat overlaps between different input nucleic acids used in an assemblyreaction can have different lengths.

EXAMPLES

Universal Polymer Array for Polymer Synthesis

Currently there is great interest in synthesizing DNA polymers which arelonger than molecules typically classified as oligomers. These polymersmay typically be of the length of 100 bases to 3000 nucleotide bases orlonger. In the current art, these longer DNA polymers are constructedfrom shorter oligonucleotides. In this example, devices and methods forconstructing an arbitrary DNA polymer sequence starting with a universaloligomer array are illustrated.

Referring to FIG. 3, a DNA oligomer array is shown in FIG. 3 a. P×Qblocks (individual solid supports) are illustrated in which each blockhas m×n oligonucleotide spots or features (see FIG. 3 b). Using atypical array fabrication methodology (e.g. ink jet depositedphosphoramidite chemistries), each spot has on the order of 10⁸oligonucleotides and may consist of oligonucleotides having a lengthless than 150 nucleosides. Such an array can also be fabricated byspotting oligonucleotides which have been synthesized off of the surfaceof the array.

FIG. 4A illustrates a construction single-stranded DNA which binds to anoligonucleotide immobilized (e.g. spotted) on an array. The constructionsingle-stranded DNA has at its 3′ end a plurality of bases (n1-n6) andcan be moved (e.g., by ink jet move) to the spot of interest. Theoligonucleotide immobilized (e.g. spotted, SEQ ID NO: 2) on the arrayhas a degenerate base region of 6 bases (N) for binding of nl-n6sequence of the construction single-stranded DNA. In this example, theoligonucleotide immobilized on the array has a particular set ofadditional bases (i.e., GTACT) to the 5′ end of the degenerate baseregion. The oligonucleotide can also have k bases of linker region(e.g., poly A) at its 3′ end. FIG. 4A illustrates the perfect matchsituation where nl-n6 anneals well to the degenerate base region. Inthis situation, a polymerase binds and stabilizes the 3′ end. Fiveadditional bases that are complementary to particular set of additionalbases of the addition region (i.e., AGTAC) can then be added to the 3′end of the construction single-stranded DNA via chain extensionreaction. In an exemplary embodiment, the number of added bases is 5,and 4⁵ =1,024 separate spots (each having a different 5-mer) can berepresented in one array that contains all possible 5 mers (1,024 intotal). This corresponds to a m×n=32×32 spot configuration in FIG. 3.

Referring to FIG. 4B, it is possible for the degenerate base region tobind the construction single-stranded DNA in a region other than the 3′end (n1-n6). In this situation, the desired set of additional baseswould not be added to the 3′ end of construction single-stranded DNAeven in the presence the addition of polymerase and dNTPs, as thepolymerase does not bind. One way to preclude this situation and toincrease chain extension reactions, is to hybridize the constructionsingle-stranded DNA to the degenerate base region at a stringenttemperature that is slightly above the melting temperature. The additionof the polymerase with dNTPs causes the hybrid or duplex in FIG. 4A tobecome stabilized even under these elevated melting conditions. Withoutwishing to be bound by theory, one possible explanation is that thepolymerase quickly extends the construction single-stranded DNA in thisconfiguration while adding more base pairs and increasing the meltingtemperature.

FIG. 5 shows the additional set of bases (e.g. AGTAC) being added to theconstruction single-stranded DNA (SEQ ID NO: 3) under the activity of apolymerase and dNTPs.

FIGS. 6A, 6B, 7A, &B and 8 illustrate one embodiment for synthesizing aDNA construct from a universal oligomer array. Referring to FIG. 6A, aDNA array containing all possible M-mers in N spots is provided (eitherby in situ synthesis or by spotting of oligonucleotides). As an example,when M =10, the array can have N=4^(M)=410 (about 10⁶) spots; this isachievable using for example, current in situ DNA array fabricationtechniques. Each oligonucleotide within each spot can be designed toinclude a universal primer binding site P′_(d) and an M-mer payload(e.g., Oligo1', Oligo2′. . . OligoN'). Referring to FIG. 6B, at step 1,a universal digestable primer P_(d) (e.g., containing cleavable uracylgroups) is introduced and hybridized to the universal priming sitesP′_(d) on the array.

Referring to FIG. 7A, at step 2, the primers P_(d) are extended using apolymerase and dNTPs, thereby producing P_(d)-Oligo1, P_(d)-Oligo2,P_(d)-Oligo 3, etc. The polymerase can contain a 3′->5′ exonucleaseactivity so as to generate a blunt ended extension product. FIG. 7Billustrates step 3 where the digestable primers P_(d) are exposed touracyl deglycosilase, digested and washed away, leaving only theconstruction oligonucleotides Oligo1, Oligo2, Oligo3, etc. . . thatremain hyrbridized at their corresponding spots.

In various embodiments, steps 1-3 can be repeated multiple times. Incertain embodiments, any intermediate products can also serve as aprimer for further chain extension reactions, thereby producing largerpolymers.

FIG. 8 illustrates an DNA array being processed by microfluidic andlaser devices. The processed DNA array can be placed in a fluidic ormicrofluidic channel which has a transparent port. Fluid can be flowedor pulsed through the channel and a laser can be used to selectivelyheat spots thus releasing the corresponding constructionoligonucleotides. In this process, the set of construction nucleotidesrequired to build a given DNA sequence by means of standard PCR assemblycan be released from the chip into a common pool to carry out a PCRassembly process. The DNA array can also be regenerated by following thesteps 1-3 of FIGS. 6 and 7 respectively.

EQUIVALENTS

The present invention provides among other things devices and methodsfor polynucleotide synthesis. While specific embodiments of the subjectinvention have been discussed, the above specification is illustrativeand not restrictive. Many variations of the invention will becomeapparent to those skilled in the art upon review of this specification.The full scope of the invention should be determined by reference to theclaims, along with their full scope of equivalents, and thespecification, along with such variations.

Incorporation By Reference

Reference is made to PCT application PCT/US09/55267, to U.S. ProvisionalApplication Ser. No. 61/257,591 filed Nov. 3, 2009, to U.S. ProvisionalApplication Ser. No. 61/264,643, entitled “Methods and Apparatus forChip based DNA error correction”, filed on Nov. 25, 2009, U.S.Provisional Application Ser. No. 61/264,632, entitled “Microfluidicdevices and methods for gene synthesis”, filed on Nov. 25, 2009, U.S.Provisional Application Ser. No. 61/264,641 entitled “Methods andDevices for the Manipulation of proplets in High Fidelity PolynucleotideAssembly”, filed Nov. 25, 2009, U.S. Provisional Application Ser. No.61/293,192, entitled “Assembly of high fidelity Polynucleotides”, filedJan. 7, 2010, U.S. Provisional Application Ser. No. 61/310,076 entitled“Assembly of high fidelity Polynucleotides”, filed on Mar. 3, 2010 andU.S. Provisional Application Ser. No. 61/310,069, entitled “Methods andMicrofluidic Devices for the Manipulation of proplets in High FidelityPolynucleotide Assembly”, filed Mar. 3, 2010. All publications, patentsand sequence database entries mentioned herein are hereby incorporatedby reference in their entirety as if each individual publication orpatent was specifically and individually indicated to be incorporated byreference.

The invention claimed is:
 1. A method for synthesizing at least one polynucleotide having a predetermined sequence, the method comprising: (a) providing a solid support having a plurality of spots thereon, wherein each of the plurality of spots comprises a plurality of oligonucleotides, each plurality of oligonucleotides having a different predetermined subunit sequence at its 5′ end and a degenerate binding sequence at its 3′ end, wherein the plurality of oligonucleotides is covalently linked at the 3′ end via a plurality of binding sequences to the solid support and wherein the predetermined subunit sequences together comprise the at least one polynucleotide; (b) providing a droplet to a first predetermined spot, the first spot comprising a first plurality of oligonucleotides having a degenerate binding sequence and a first predetermined subunit sequence, wherein the droplet comprises a sequence capable of annealing to the degenerate sequence of the first plurality of oligonucleotides and one or more reagents that allow one or more of annealing, denaturing, chain extension, ligation, and digestion reaction to produce a first extension product comprising the first predetermined subunit sequence; (c) advancing a droplet comprising the first extension product from the first spot to a predetermined second spot, the second spot having a second plurality of oligonucleotides comprising a degenerate binding sequence and a second predetermined subunit sequence different than the first predetermined sequence; and (d) providing one or more reagents thereby allowing one or more of annealing, denaturing, chain extension, ligation, and digestion reaction at the second spot to produce a second extension product comprising the first and the second predetermined subunit sequences.
 2. The method of claim 1 wherein the degenerate sequences have a length of N1 nucleosides, wherein the plurality of binding sequences comprises up to 4^(N1) different sequences and wherein N1 is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
 20. 3. The method of claim 1 wherein the subunit sequence has a length of N2 nucleosides and is one of 4^(N2) possible sequences, wherein the solid support comprises 4^(N2) spots or a subset or superset thereof and wherein N2 is 3, 4, 5, 6, 7, 8, 9, or
 10. 4. The method of claim 1, further comprising individually controlling a temperature of the droplet at each of the plurality of spots on the solid support, wherein the temperature is controlled at 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C. or 20° C. above a predetermined temperature which corresponds to an average annealing temperature of the plurality of binding sequences.
 5. The method of claim 4 wherein the temperature is controlled at above the predetermined temperature such that duplexes stabilized by a polymerase or a subunit thereof remain substantially annealed at said temperature.
 6. The method of claim 1 further comprising providing in the droplet a primer that at least partially binds to at least one of the plurality of the binding sequences on the first spot, a polymerase or a subunit thereof, and dNTPs, thereby allowing chain extension of the primer using the first predetermined subunit sequence as a template to produce the first extension product comprising the first predetermined subunit sequence.
 7. The method of claim 6 further comprising cleaving the primer to remove unwanted primer sequences from the extension product comprising the predetermined sequence.
 8. The method of claim 6 further comprising using the first extension product as a primer for chain extension at the second spot comprising the second plurality of oligonucleotides having the degenerate binding sequence and the second predetermined subunit sequence and using the second predetermined subunit sequence as a template to produce the second extension product comprising the first and the second predetermined subunit sequences.
 9. The method of claim 1 wherein the droplet is advanced by electrical, magnetic or mechanical action.
 10. The method of claim 1 wherein a predetermined combination of a subset of spots is used to produce the at least one polynucleotide.
 11. The method of claim 1 wherein the degenerate sequence is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides long.
 12. The method of claim 1 further repeating steps c and d to produce the at least one polynucleotide having the predetermined sequence.
 13. A method for synthesizing at least one polynucleotide having a predetermined sequence, the method comprising: (a) providing a solid support having a plurality of distinct features thereon, each feature comprising a plurality of oligonucleotides bound to the support through their 3′ end, each oligonucleotide having a sequence complementary to a predetermined subunit sequence at its 5′ end and a degenerate binding sequence at its 3′ end, wherein the predetermined subunit sequences together comprise the at least one polynucleotide; (b) providing a droplet to a first predetermined feature comprising a first plurality of oligonucleotides, wherein the droplet comprises a sequence capable of annealing to the degenerate sequence of the first plurality of oligonucleotides; (c) providing one or more reagents thereby allowing one or more of annealing, denaturing, chain extension, ligation, and digestion reaction; (d) generating within a droplet a first extension product comprising the first predetermined subunit sequence; (e) advancing the droplet comprising the first extension product from the first feature to a predetermined second feature, the second feature having a second plurality of oligonucleotides comprising a degenerate binding sequence and a sequence complementary to a second predetermined subunit sequence different than the first predetermined sequence; (f) providing one or more reagents thereby allowing one or more of annealing, denaturing, chain extension, ligation, and digestion reaction at the second feature; and (h) generating a second extension product comprising the first and the second predetermined subunit sequences. 